Would Near-infrared Fluorescence Signals Propagate Through Large Human Organs for Clinical Studies?

Optics Letters. Mar, 2002  |  Pubmed ID: 18007794

We predict the capacity of near-infrared fluorescent signals to propagate through human tissue for non-invasive medical imaging. This analysis employs experimental measurements of a biologically relevant local fluorochrome embedded in tissuelike media and predicts the equivalent photon counts expected from breast, lung, brain, and muscle as a function of diameter by use of an analytical solution of the diffusion equation that can take into account large arbitrary geometries. The findings address feasibility issues for clinical studies and are relevant to recent development of near-infrared fluorescent probes and molecular beacons for in vivo applications.

Fast Analytical Approximation for Arbitrary Geometries in Diffuse Optical Tomography

Optics Letters. Apr, 2002  |  Pubmed ID: 18007854

Diffuse optical tomography is a novel imaging technique that resolves and quantifies the optical properties of objects buried in turbid media. Typically, numerical solutions of the diffusion equation are employed to construct the tomographic problem when media of complex geometries are investigated. Numerical methods offer implementation simplicity but also significant computation burden, especially when large three-dimensional reconstructions are involved. We present an alternative method of performing tomography of diffuse media of arbitrary geometries by means of an analytical approach, the Kirchhoff approximation. We show that the method is extremely efficient in computation times and consider its potential as a real-time three-dimensional imaging tool.

Would Near-infrared Fluorescence Signals Propagate Through Large Human Organs for Clinical Studies? Errata

Optics Letters. Sep, 2002  |  Pubmed ID: 18026531

Osteogenesis Associates with Inflammation in Early-stage Atherosclerosis Evaluated by Molecular Imaging in Vivo

Circulation. Dec, 2007  |  Pubmed ID: 18040026

Arterial calcification is associated with cardiovascular events; however, mechanisms of calcification in atherosclerosis remain obscure.

Effect of Tumor Microenvironment Modulation on the Efficacy of Oncolytic Virus Therapy

Journal of the National Cancer Institute. Dec, 2007  |  Pubmed ID: 18042934

The tumor microenvironment is being increasingly recognized as an important determinant of tumor progression as well as of therapeutic response. We investigated oncolytic virus (OV) therapy-induced changes in tumor blood vessels and the impact of modulating tumor vasculature on the efficacy of oncolytic virus therapy.

A Fluorescent Probe for the Detection of Myeloperoxidase Activity in Atherosclerosis-associated Macrophages

Chemistry & Biology. Nov, 2007  |  Pubmed ID: 18022561

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl/OCl(-)) is implicated in the pathogenesis of atherosclerosis and other inflammatory states. We have synthesized an imaging probe, sulfonaphthoaminophenyl fluorescein (SNAPF), that selectively reacts with HOCl. SNAPF detects HOCl produced by stimulated MPO-expressing cells cultured from human whole blood, as well as HOCl from bone marrow (BM)-derived macrophages isolated from transgenic mice that express human MPO. Two lines of evidence indicate that SNAPF permits the in vivo imaging of HOCl production. First, we used this approach to demonstrate HOCl production by neutrophils in experimental murine peritonitis. Second, we detected HOCl production by MPO expressing cells in human atherosclerotic arteries. Thus, fluorescence reflectance imaging by SNAPF may provide a valuable noninvasive molecular imaging tool for implicating HOCl and MPO in the damage of inflamed tissues.

The Healing Myocardium Sequentially Mobilizes Two Monocyte Subsets with Divergent and Complementary Functions

The Journal of Experimental Medicine. Nov, 2007  |  Pubmed ID: 18025128

Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6C(hi) and -6C(lo) monocytes via CCR2 and CX(3)CR1, respectively. Ly-6C(hi) monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6C(lo) monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular-endothelial growth factor. Consequently, Ly-6C(hi) monocytes digest damaged tissue, whereas Ly-6C(lo) monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6C(hi) monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.

Mast Cells Are an Essential Hematopoietic Component for Polyp Development

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2007  |  Pubmed ID: 18077429

It is generally agreed that most colon cancers develop from adenomatous polyps, and it is this fact on which screening strategies are based. Although there is overwhelming evidence to link intrinsic genetic lesions with the formation of these preneoplastic lesions, recent data suggest that the tumor stromal environment also plays an essential role in this disease. In particular, it has been suggested that CD34(+) immature myeloid precursor cells are required for tumor development and invasion. Here we have used mice conditional for the stabilization of beta-catenin or defective for the adenomatous polyposis coli (APC) gene to reinvestigated the identity and importance of tumor-infiltrating hematopoietic cells in polyposis. We show that, from the onset, polyps are infiltrated with proinflammatory mast cells (MC) and their precursors. Depletion of MC either pharmacologically or through the generation of chimeric mice with genetic lesions in MC development leads to a profound remission of existing polyps. Our data suggest that MC are an essential hematopoietic component for preneoplastic polyp development and are a novel target for therapeutic intervention.

Utility of a New Bolus-injectable Nanoparticle for Clinical Cancer Staging

Neoplasia (New York, N.Y.). Dec, 2007  |  Pubmed ID: 18084623

In this study, we report on the use of a new, bolus-injectable, carboxymethyl dextran-based magnetic nanoparticle (MNP), ferumoxytol, to improve detection in loco-regional lymph nodes by magnetic resonance imaging (MRI).

Nanoparticle PET-CT Imaging of Macrophages in Inflammatory Atherosclerosis

Circulation. Jan, 2008  |  Pubmed ID: 18158358

Macrophages participate centrally in atherosclerosis, and macrophage markers (eg, CD68, MAC-3) correlate well with lesion severity and therapeutic modulation. On the basis of the avidity of lesional macrophages for polysaccharide-containing supramolecular structures such as nanoparticles, we have developed a new positron emission tomography (PET) agent with optimized pharmacokinetics to allow in vivo imaging at tracer concentrations.

Noninvasive in Vivo Imaging of Monocyte Trafficking to Atherosclerotic Lesions

Circulation. Jan, 2008  |  Pubmed ID: 18172031

Monocytes play a key role in atherogenesis, but their participation has been discerned largely via ex vivo analyses of atherosclerotic lesions. We sought to establish a noninvasive technique to determine monocyte trafficking to atherosclerotic lesions in live animals.

A Secreted Luciferase for Ex Vivo Monitoring of in Vivo Processes

Nature Methods. Feb, 2008  |  Pubmed ID: 18204457

Luciferases are widely used to monitor biological processes. Here we describe the naturally secreted Gaussia princeps luciferase (Gluc) as a highly sensitive reporter for quantitative assessment of cells in vivo by measuring its concentration in blood. The Gluc blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models.

Myeloperoxidase-targeted Imaging of Active Inflammatory Lesions in Murine Experimental Autoimmune Encephalomyelitis

Brain : a Journal of Neurology. Apr, 2008  |  Pubmed ID: 18234693

Inflammatory demyelinating plaques are the pathologic hallmark of active multiple sclerosis and often precede clinical manifestations. Non-invasive early detection of active plaques would thus be crucial in establishing pre-symptomatic diagnosis and could lead to early preventive treatment strategies. Using murine experimental autoimmune encephalomyelitis as a model of multiple sclerosis, we demonstrate that a prototype paramagnetic myeloperoxidase (MPO) sensor can detect and confirm more, smaller, and earlier active inflammatory lesions in living mice by in vivo MRI. We show that MPO expression corresponded with areas of inflammatory cell infiltration and demyelination, and higher MPO activity as detected by MPO imaging, biochemical assays, and histopathological analyses correlated with increased clinical disease severity. Our findings present a potential new translational approach for specific non-invasive inflammatory plaque imaging. This approach could be used in longitudinal studies to identify active demyelinating plaques as well as to more accurately track disease course following treatment in clinical trials.

Activatable Magnetic Resonance Imaging Agent Reports Myeloperoxidase Activity in Healing Infarcts and Noninvasively Detects the Antiinflammatory Effects of Atorvastatin on Ischemia-reperfusion Injury

Circulation. Mar, 2008  |  Pubmed ID: 18268141

Ischemic injury of the myocardium causes timed recruitment of neutrophils and monocytes/macrophages, which produce substantial amounts of local myeloperoxidase (MPO). MPO forms reactive chlorinating species capable of inflicting oxidative stress and altering protein function by covalent modification. We have used a small-molecule, gadolinium-based activatable sensor for magnetic resonance imaging of MPO activity (MPO-Gd). MPO-Gd is first radicalized by MPO and then either spontaneously oligomerizes or binds to matrix proteins, all leading to enhanced spin-lattice relaxivity and delayed washout kinetics. We hypothesized that MPO imaging could be used to measure inflammatory responses after myocardial ischemia locally and noninvasively in a murine model.

Transglutaminase Activity in Acute Infarcts Predicts Healing Outcome and Left Ventricular Remodelling: Implications for FXIII Therapy and Antithrombin Use in Myocardial Infarction

European Heart Journal. Feb, 2008  |  Pubmed ID: 18276618

The transglutaminase factor XIII (FXIII) emerges as a key enzyme in healing after myocardial infarction (MI). Here we assess the impact of transglutaminase-modulating therapies on healing and evolution of heart failure using a novel, non-invasive molecular imaging technique.

A Novel Method of Imaging Calcium Urolithiasis Using Fluorescence

The Journal of Urology. Apr, 2008  |  Pubmed ID: 18295253

In the surgical management of urolithiasis the goal of treatment is not only to remove calculi, but also prevent future stone formation by rendering the patient stone-free/fragment-free. Achieving this goal is often difficult with endoscopic procedures due to the inability to visualize small calculi well even with x-ray or ultrasound. We evaluated fluorescence probes as a novel method of identifying calculi in the urinary tract.

Pilot Study Evaluating Use of Lymphotrophic Nanoparticle-enhanced Magnetic Resonance Imaging for Assessing Lymph Nodes in Renal Cell Cancer

Urology. Apr, 2008  |  Pubmed ID: 18295316

To assess lymphotrophic nanoparticle-enhanced magnetic resonance imaging (LNMRI) in identifying malignant nodal involvement in patients with renal neoplasms.

Magnetic Microparticle Aggregation for Viscosity Determination by MR

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Mar, 2008  |  Pubmed ID: 18306403

Micron-sized magnetic particles were induced to aggregate when placed in homogeneous magnetic fields, like those of MR imagers and relaxometers, and then spontaneously returned to their dispersed state when removed from the field. Associated with the aggregation and dispersion of the magnetic particles were time-dependent increases and decreases in the spin-spin relaxation time (T2) of the water. Magnetic nanoparticles, with far smaller magnetic moments per particle, did not undergo magnetically induced aggregation and exhibited time-independent values of T2. The rate of T2 change associated with magnetic microparticle aggregation was used to determine the viscosity of liquid samples, providing a method that can be of particular advantage for determining the viscosity of small volumes of potentially biohazardous samples of blood or blood plasma.

MR-optical Imaging of Cardiovascular Molecular Targets

Basic Research in Cardiology. Mar, 2008  |  Pubmed ID: 18324364

Our understanding of the intricate inflammation biology underlying atherosclerosis is rapidly progressing. Molecular imaging strategies, harnessing this body of knowledge, have been developed to visualize some key cellular and molecular events in plaque evolution and vulnerability. Here, we discuss recent advances in magnetic resonance and fluorescence imaging of key biomarkers including adhesion molecules, inflammatory cells, and enzyme activity. We discuss strengths and limitations of respective imaging technologies, and comment on the potential of multi-modality imaging approaches.

Magnetic Nanoparticles for MR Imaging: Agents, Techniques and Cardiovascular Applications

Basic Research in Cardiology. Mar, 2008  |  Pubmed ID: 18324368

Magnetic nanoparticles (MNP) are playing an increasingly important role in cardiovascular molecular imaging. These agents are superparamagnetic and consist of a central core of iron-oxide surrounded by a carbohydrate or polymer coat. The size, physical properties and pharmacokinetics of MNP make them highly suited to cellular and molecular imaging of atherosclerotic plaque and myocardial injury. MNP have a sensitivity in the nanomolar range and can be detected with T1, T2, T2*, off resonance and steady state free precession sequences. Targeted imaging with MNP is being actively explored and can be achieved through either surface modification or through the attachment of an affinity ligand to the nanoparticle. First generation MNP are already in clinical use and second generation agents, with longer blood half lives, are likely to be approved for routine clinical use in the near future.

Detection of Early Prostate Cancer Using a Hepsin-targeted Imaging Agent

Cancer Research. Apr, 2008  |  Pubmed ID: 18381435

Early detection and diagnosis of prostate cancer is key to designing effective treatment strategies. Microarrays have resulted in the discovery of hepsin (HPN) as a biomarker for detection of prostate cancer. In this study, we explore the development of HPN imaging probes for detection of prostate cancer. We used phage display to isolate HPN binding peptides with 190 + 2.2 nmol/L affinity in monomeric form and high specificity. The identified peptides were able to detect human prostate cancer on tissue microarrays and in cell-based assays. HPN-targeted imaging agents were synthesized by conjugating multiple peptides to fluorescent nanoparticles to further improve avidity through multivalency and to improve pharmacokinetics. When injected into mouse xenograft models, HPN-targeted nanoparticles bound specifically to HPN-expressing LNCaP xenografts compared with non-HPN-expressing PC3 xenografts. HPN imaging may provide a new method for detection of prostate cancer.

Imaging in the Era of Molecular Oncology

Nature. Apr, 2008  |  Pubmed ID: 18385732

New technologies for imaging molecules, particularly optical technologies, are increasingly being used to understand the complexity, diversity and in vivo behaviour of cancers. 'Omic' approaches are providing comprehensive 'snapshots' of biological indicators, or biomarkers, of cancer, but imaging can take this information a step further, showing the activity of these markers in vivo and how their location changes over time. Advances in experimental and clinical imaging are likely to improve how cancer is understood at a systems level and, ultimately, should enable doctors not only to locate tumours but also to assess the activity of the biological processes within these tumours and to provide 'on the spot' treatment.

Targeted Nanoparticles for Imaging Incipient Pancreatic Ductal Adenocarcinoma

PLoS Medicine. Apr, 2008  |  Pubmed ID: 18416599

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.

Sensitive NMR Sensors Detect Antibodies to Influenza

Angewandte Chemie (International Ed. in English). 2008  |  Pubmed ID: 18428168

Bimodal Viral Vectors and in Vivo Imaging Reveal the Fate of Human Neural Stem Cells in Experimental Glioma Model

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2008  |  Pubmed ID: 18434519

Transplantation of genetically engineered cells into the CNS offers immense potential for the treatment of several neurological disorders. Monitoring expression levels of transgenes and following changes in cell function and distribution over time is critical in assessing therapeutic efficacy of such cells in vivo. We have engineered lentiviral vectors bearing fusions between different combinations of fluorescent and bioluminescent marker proteins and used bioluminescence imaging and intravital-scanning microscopy in real time to study the fate of human neural stem cells (hNSCs) at a cellular resolution in glioma-bearing brains in vivo. Using Renilla luciferase (Rluc)-DsRed2 or GFP-Rluc-expressing malignant human glioma model, transduced hNSCs were shown to migrate extensively toward gliomas, with hNSCs populating gliomas at 10 d after transplantation. Furthermore, transduced hNSCs survived longer in mice with gliomas than in normal brain, but did not modulate glioma progression in vivo. These studies demonstrate the utility of bimodal viral vectors and real-time imaging in evaluating fate of NSCs in diseased models and thus provide a platform for accelerating cell-based therapies for CNS disorders.

Perturbational Profiling of Nanomaterial Biologic Activity

Proceedings of the National Academy of Sciences of the United States of America. May, 2008  |  Pubmed ID: 18492802

Our understanding of the biologic effects (including toxicity) of nanomaterials is incomplete. In vivo animal studies remain the gold standard; however, widespread testing remains impractical, and the development of in vitro assays that correlate with in vivo activity has proven challenging. Here, we demonstrate the feasibility of analyzing in vitro nanomaterial activity in a generalizable, systematic fashion. We assessed nanoparticle effects in a multidimensional manner, using multiple cell types and multiple assays that reflect different aspects of cellular physiology. Hierarchical clustering of these data identifies nanomaterials with similar patterns of biologic activity across a broad sampling of cellular contexts, as opposed to extrapolating from results of a single in vitro assay. We show that this approach yields robust and detailed structure-activity relationships. Furthermore, a subset of nanoparticles were tested in mice, and nanoparticles with similar activity profiles in vitro exert similar effects on monocyte number in vivo. These data suggest a strategy of multidimensional characterization of nanomaterials in vitro that can inform the design of novel nanomaterials and guide studies of in vivo activity.

Multifunctional Magnetic Nanoparticles for Targeted Imaging and Therapy

Advanced Drug Delivery Reviews. Aug, 2008  |  Pubmed ID: 18508157

Magnetic nanoparticles have become important tools for the imaging of prevalent diseases, such as cancer, atherosclerosis, diabetes, and others. While first generation nanoparticles were fairly nonspecific, newer generations have been targeted to specific cell types and molecular targets via affinity ligands. Commonly, these ligands emerge from phage or small molecule screens, or are based on antibodies or aptamers. Secondary reporters and combined therapeutic molecules have further opened potential clinical applications of these materials. This review summarizes some of the recent biomedical applications of these newer magnetic nanomaterials.

Electrode Chemistry Yields a Nanoparticle-based NMR Sensor for Calcium

Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2008  |  Pubmed ID: 18558729

Magnetic nanoparticles (NPs) have been used to obtain NMR-based sensors for analytes ranging from small molecules to viruses by the conjugation of biomolecules (antibodies, proteins, oligonucleotides) to the surface of NPs. In the presence of an analyte, the NPs form clusters that alter the relaxation time of the surrounding water protons. Here, we show that an organic molecule that binds calcium ions of nonbiological origin, rather than a biomolecule, can be employed to modify the surface of a magnetic NP. When calcium ions are added, they induce NP clustering, providing an NMR-based sensor for these ions. Our work suggests that the many chemistries of nonbiological origin, such as those employed for ion-selective electrodes, can be adapted to obtain NMR-based sensors for ions.

Chip-NMR Biosensor for Detection and Molecular Analysis of Cells

Nature Medicine. Aug, 2008  |  Pubmed ID: 18607350

Rapid and accurate measurement of biomarkers in tissue and fluid samples is a major challenge in medicine. Here we report the development of a new, miniaturized diagnostic magnetic resonance (DMR) system for multiplexed, quantitative and rapid analysis. By using magnetic particles as a proximity sensor to amplify molecular interactions, the handheld DMR system can perform measurements on unprocessed biological samples. We show the capability of the DMR system by using it to detect bacteria with high sensitivity, identify small numbers of cells and analyze them on a molecular level in real time, and measure a series of protein biomarkers in parallel. The DMR technology shows promise as a robust and portable diagnostic device.

Slow Self-activation Enhances the Potency of Viridin Prodrugs

Journal of Medicinal Chemistry. Aug, 2008  |  Pubmed ID: 18630894

When the viridin wortmannin (Wm) is modified by reaction with certain nucleophiles at the C20 position, the compounds obtained exhibit an improved antiproliferative activity even though a covalent reaction between C20 and a lysine in the active site of PI3 kinase is essential to Wm's ability to inhibit this enzyme. Here we show that this improved potency results from an intramolecular attack by the C6 hydroxyl group that slowly converts these inactive prodrugs to the active species Wm over the 48 h duration of the antiproliferative assay. Our results provide a guide for selecting Wm-like compounds to maximize kinase inhibition with the variety of protocols used to assess the role of PI3 kinase in biological systems, or for achieving optimal therapeutic effects in vivo . In addition, the slow self-activation of WmC20 derivatives provides a mechanism that can be exploited to obtain kinase inhibitors endowed with physical and pharmacokinetic properties far different from man-made kinase inhibitors because they do not bind to kinase active sites.

In Vivo Quantitative Microvasculature Phenotype Imaging of Healthy and Malignant Tissues Using a Fiber-optic Confocal Laser Microprobe

Translational Oncology. Jul, 2008  |  Pubmed ID: 18633456

Real-time in vivo imaging of the microvasculature may help both earlier clinical detection of disease and the understanding of tumor-host interaction at various stages of progression. In vivo confocal and multiphoton microscopy is often hampered by bulky optics setup and has limited access to internal organs. A fiber-optic setup avoids these limitations and offers great user maneuverability. We report here the in vivo validation of a fiber-optic confocal fluorescence microprobe imaging system. In addition, we developed an automated fractal-based image analysis to characterize microvascular morphology based on vessel diameter distribution, density, volume fraction, and fractal dimension from real-time data. The system is optimized for use in the far-red and near-infrared region. The flexible 1.5-mm-diameter fiber-optic bundle and microprobe enable great user maneuverability, with a field of view of 423 x 423 microm and a tissue penetration of up to 15 microm. Lateral and axial resolutions are 3.5 and 15 microm. We show that it is possible to obtain high temporal and spatial resolution images of virtually any abdominal viscera in situ using a far-red blood pool imaging probe. Using an orthotopic model of pancreatic ductal adenocarcinoma, we characterized the tumor surface capillary and demonstrated that the imaging system and analysis can quantitatively differentiate between the normal and tumor surface capillary. This clinically approved fiber-optic system, together with the fractal-based image analysis, can potentially be applied to characterize other tumors in vivo and may be a valuable tool to facilitate their clinical evaluation.

Targeted Imaging of Myocardial Damage

Nature Clinical Practice. Cardiovascular Medicine. Aug, 2008  |  Pubmed ID: 18641609

Molecular imaging agents can be targeted to a specific receptor or protein on the cardiomyocyte surface, or to enzymes released into the interstitial space, such as cathepsins, matrix metalloproteinases and myeloperoxidase. Molecular imaging of the myocardium, however, requires the imaging agent to be small, sensitive (nanomolar levels or better), and able to gain access to the interstitial space. Several novel agents that fulfill these criteria have been used for targeted molecular imaging applications in the myocardium. Magnetic resonance, fluorescence, and single-photon emission CT have been used to image the molecular signals generated by these agents. The use of targeted imaging agents in the myocardium has the potential to provide valuable insights into the pathophysiology of myocardial injury and to facilitate the development of novel therapeutic strategies.

Human Breast Cancer Tumor Models: Molecular Imaging of Drug Susceptibility and Dosing During HER2/neu-targeted Therapy

Radiology. Sep, 2008  |  Pubmed ID: 18647846

To use near-infrared (NIR) optical imaging to assess the therapeutic susceptibility and drug dosing of orthotopic human breast cancers implanted in mice treated with molecularly targeted therapy.

Near Infrared Fluorescence-based Bacteriophage Particles for Ratiometric PH Imaging

Bioconjugate Chemistry. Aug, 2008  |  Pubmed ID: 18666791

Fluorogenic imaging agents emitting in the near-infrared are becoming important research tools for disease investigation in vivo. Often pathophysiological states such as cancer and cystic fibrosis are associated with disruptions in acid/base homeostasis. The development of optical sensors for pH imaging would facilitate the investigation of these diseased conditions. In this report, the design and synthesis of a ratiometric near-infrared emitting probe for pH quantification is detailed. The pH-responsive probe is prepared by covalent attachment of pH-sensitive and pH-insensitive fluorophores to a bacteriophage particle scaffold. The pH-responsive cyanine dye, HCyC-646, used to construct the probe, has a fluorogenic pKa of 6.2, which is optimized for visualization of acidic pH often associated with tumor hypoxia and other diseased states. Incorporation of pH-insensitive reference dyes enables the ratiometric determination of pH independent of the probe concentration. With the pH-responsive construct, measurement of intracellular pH and accurate determination of pH through optically diffuse biological tissue is demonstrated.

Noninvasive Detection of Macrophage-rich Atherosclerotic Plaque in Hyperlipidemic Rabbits Using "positive Contrast" Magnetic Resonance Imaging

Journal of the American College of Cardiology. Aug, 2008  |  Pubmed ID: 18672170

This study was designed to identify macrophage-rich atherosclerotic plaque noninvasively by imaging the tissue uptake of long-circulating superparamagnetic nanoparticles with a positive contrast off-resonance imaging sequence (inversion recovery with ON-resonant water suppression [IRON]).

Live Imaging of Cysteine-cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

PloS One. 2008  |  Pubmed ID: 18698347

It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR) fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC) gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4) of the probe signal was localized in CD11b(+)Gr1(+) myeloid derived suppressor cells (MDSC) and CD11b(+)F4/80(+) macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.

Off-resonance Angiography: a New Method to Depict Vessels--phantom and Rabbit Studies

Radiology. Nov, 2008  |  Pubmed ID: 18780823

To evaluate the utility of inversion recovery with on-resonant water suppression (IRON) in combination with injection of the long-circulating monocrystalline iron oxide nanoparticle (MION)-47 for contrast material-enhanced magnetic resonance (MR) angiography. MATERIALS AND METhods: Experiments were approved by the institutional animal care committee. Eleven rabbits were imaged at baseline before injection of a contrast agent and then serially 5-30 minutes, 2 hours, 1 day, and 3 days after a single intravenous bolus injection of 80 micromol of MION-47 per kilogram of body weight (n = 6) or 250 micromol/kg MION-47 (n = 5). Conventional T1-weighted MR angiography and IRON MR angiography were performed on a clinical 3.0-T imager. Signal-to-noise and contrast-to-noise ratios were measured in the aorta of rabbits in vivo. Venous blood was obtained from the rabbits before and after MION-47 injection for use in phantom studies.

Integrated Nanosensors to Determine Levels and Functional Activity of Human Telomerase

Neoplasia (New York, N.Y.). Oct, 2008  |  Pubmed ID: 18813356

Telomerase is a key oncogenic enzyme, and a number of novel telomerase inhibitors are currently under development. Because inhibition can be achieved either at the protein or at the enzymatic activity level, independent measurements of these parameters are important in the development of effective therapeutic agents. In the current study, we have developed a set of functional magnetic nanosensors capable of measuring the concentration of telomerase, as well as its enzymatic activity in parallel. The method is based on a magnetic relaxation switch assay, which can be performed in crude tissue samples and is fast and extremely sensitive. Using this method, we were able to detect different amounts of telomerase protein and activity in various cancer and normal cell lines. Furthermore, we were able to study the effect of phosphorylation on telomerase activity. This system not only could provide a rapid assay for the evaluation of antitelomerase therapies but could also be implemented to the study of other cancer markers.

Simplified Syntheses of Complex Multifunctional Nanomaterials

Chemical Communications (Cambridge, England). Oct, 2008  |  Pubmed ID: 18830495

Multifunctional probes are synthesized in a single step using peptide scaffold-based multifunctional single-attachment-point (MSAP) reagents.

Real-time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis

Circulation. Oct, 2008  |  Pubmed ID: 18852366

To enable intravascular detection of inflammation in atherosclerosis, we developed a near-infrared fluorescence (NIRF) catheter-based strategy to sense cysteine protease activity during vascular catheterization.

Regression of Drug-resistant Lung Cancer by the Combination of Rosiglitazone and Carboplatin

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2008  |  Pubmed ID: 18927287

Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARgamma ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARgamma/carboplatin combination in these more relevant models of drug resistant non-small cell lung cancer.

Magnetic Resonance Imaging Monitors Physiological Changes with Antihedgehog Therapy in Pancreatic Adenocarcinoma Xenograft Model

Pancreas. Nov, 2008  |  Pubmed ID: 18953259

The sonic hedgehog (Shh) pathway has an established role in pancreatic cancer (pancreatic adenocarcinoma [PDAC]). We tested whether magnetic resonance imaging measures of vascular volume fraction (VVF) using magnetic iron oxide nanoparticles are sensitive to the antiangiogenic effect of targeted Shh therapies in a PDAC xenograft model.

MiR-296 Regulates Growth Factor Receptor Overexpression in Angiogenic Endothelial Cells

Cancer Cell. Nov, 2008  |  Pubmed ID: 18977327

A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here, we demonstrate that a small regulatory microRNA, miR-296, has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.

Targeting Multiple Pathways in Gliomas with Stem Cell and Viral Delivered S-TRAIL and Temozolomide

Molecular Cancer Therapeutics. Nov, 2008  |  Pubmed ID: 19001440

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells. However, its short half-life, poor delivery, and TRAIL-resistant tumor cells have diminished its clinical efficacy. In this study, we explored whether novel delivery methods will represent new and effective ways to treat gliomas and if adjuvant therapy with the chemotherapeutic agent temozolomide would enhance the cytotoxic properties of TRAIL in glioma lines resistant to TRAIL monotherapy. We have engineered adeno-associated virus (AAV) vectors encoding recombinant secreted TRAIL (S-TRAIL) and bioluminescent-fluorescent marker fusion proteins and show that AAV-delivered S-TRAIL leads to varying degrees of killing in multiple glioma lines, which correspond with caspase-3/7 activation. In vivo, dual bioluminescent imaging revealed efficient delivery of therapeutic AAV vectors directly into the tumor mass, which induced marked attenuation of tumor progression. Treatment of glioma cells with the chemotherapeutic agent temozolomide alone lead to a significant accumulation of cells in G(2)-M phase, activated the cell cycle checkpoint protein Chk1, and increased death receptor expression in a time-dependent manner. Furthermore, combined treatment with AAV-S-TRAIL or neural stem cell-S-TRAIL and temozolomide induced cell killing and markedly up-regulated proapoptotic proteins in glioma cells least sensitive to TRAIL. This study elucidates novel means of delivering S-TRAIL to gliomas and suggests combination of clinically relevant temozolomide and S-TRAIL may represent a new therapeutic option with increased potency for glioblastoma patients.

Notch Signaling in Cardiovascular Disease and Calcification

Current Cardiology Reviews. Aug, 2008  |  Pubmed ID: 19936191

Recent increase in human lifespan has shifted the spectrum of aging-related disorders to an unprecedented upsurge in cardiovascular diseases, especially calcific aortic valve stenosis, which has an 80% risk of progression to heart failure and death. A current therapeutic option for calcified valves is surgical replacement, which provides only temporary relief. Recent progress in cardiovascular research has suggested that arterial and valve calcification are the result of an active process of osteogenic differentiation, induced by a pro-atherogenic inflammatory response. At molecular level, the calcification process is regulated by a network of signaling pathways, including Notch, Wnt and TGFbeta/BMP pathways, which control the master regulator of osteogenesis Cbfa1/Runx2. Genetic and in vitro studies have implicated Notch signaling in the regulation of macrophage activation and cardiovascular calcification. Individuals with inactivating Notch1 mutations have a high rate of cardiovascular disorders, including valve stenosis and calcification. This article reviews recent progress in the mechanism of cardiovascular calcification and discusses potential molecular mechanisms involved, focusing on Notch receptors. We propose a calcification model where extreme increases in vascular wall cell density due to inflammation-induced cell proliferation can trigger an osteogenic differentiation program mediated by Notch receptors.

Development of a Time Domain Fluorimeter for Fluorescent Lifetime Multiplexing Analysis

IEEE Transactions on Biomedical Circuits and Systems. Sep, 2008  |  Pubmed ID: 19830273

We show that a portable, inexpensive USB-powered time domain fluorimeter (TDF) and analysis scheme were developed for use in evaluating a new class of fluorescent lifetime multiplexed dyes. Fluorescent proteins, organic dyes, and quantum dots allow the labeling of more and more individual features within biological systems, but the wide absorption and emission spectra of these fluorophores limit the number of distinct processes which may be simultaneously imaged using spectral separation alone. By additionally separating reporters in a second dimension, fluorescent lifetime multiplexing provides a means to multiply the number of available imaging channels.

A Highly Selective Fluorescent Probe for Thiol Bioimaging

Organic Letters. Jan, 2008  |  Pubmed ID: 18062694

A new fluorescent turn-on probe (3) for the selective sensing and bioimaging of thiols is reported. In aqueous buffer solutions at physiological pH, thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the red-emissive donor-acceptor fluorophore (4). The probe displays excellent immunity to interference from nitrogen and oxygen nucleophiles and the imaging of thiols in living cells is demonstrated.

Noninvasive Imaging of Apoptosis in Cardiovascular Disease

Heart Failure Reviews. Jun, 2008  |  Pubmed ID: 18074226

Recent advances in molecular imaging have permitted the noninvasive imaging of apoptosis, a critical process underlying the pathogenesis of many diseases of the cardiovascular system including atherosclerotic vascular disease, myocardial ischemia and reperfusion injury, chronic heart failure, myocarditis, and cardiac allograft rejection. Multiple molecular targets including phosphatidylserine, phosphatidylinositol 3-kinase, and caspases have been targeted by a variety of imaging agents and modalities such as nuclear scintigraphy, PET, MRI, and fluorescent and bioluminescent imaging. Translationally, methods utilizing radiolabeled annexin V have proven promising in several clinical trials of ischemia-reperfusion injury and cardiac allograft rejection. New approaches using novel molecular imaging agents show great potential for the ability to image apoptosis in the research and clinical setting. Ultimately the ability to detect apoptosis noninvasively would help to identify patients for emerging anti-apoptotic therapies and guide clinical management with the aim of maximal myocardial preservation.

Tracking the Inflammatory Response in Stroke in Vivo by Sensing the Enzyme Myeloperoxidase

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2008  |  Pubmed ID: 19011099

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.

Early Photon Tomography Allows Fluorescence Detection of Lung Carcinomas and Disease Progression in Mice in Vivo

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2008  |  Pubmed ID: 19015534

Imaging of targeted fluorescent probes offers significant advantages for investigating disease and tissue function in animal models in vivo. Conversely, macroscopic tomographic imaging is challenging because of the high scatter of light in biological tissue and the ill-posed nature of the reconstruction mathematics. In this work, we use the earliest-transmitted photons through Lewis Lung Carcinoma bearing mice, thereby dramatically reducing the effect of tissue scattering. By using a fluorescent probe sensitive to cysteine proteases, the method yielded outstanding imaging performance compared with conventional approaches. Accurate visualization of biochemical abnormalities was achieved, not only in the primary tumor, but also in the surrounding tissue related to cancer progression and inflammatory response at the organ level. These findings were confirmed histologically and with ex vivo fluorescence microscopy. The imaging fidelity demonstrated underscores a method that can use a wide range of fluorescent probes to accurately visualize cellular- and molecular-level events in whole animals in vivo.

Effective Use of PI3K and MEK Inhibitors to Treat Mutant Kras G12D and PIK3CA H1047R Murine Lung Cancers

Nature Medicine. Dec, 2008  |  Pubmed ID: 19029981

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Real-time Assessment of Inflammation and Treatment Response in a Mouse Model of Allergic Airway Inflammation

The Journal of Clinical Investigation. Dec, 2008  |  Pubmed ID: 19033674

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Tetrazine-based Cycloadditions: Application to Pretargeted Live Cell Imaging

Bioconjugate Chemistry. Dec, 2008  |  Pubmed ID: 19053305

Bioorthogonal tetrazine cycloadditions have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aqueous media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-infrared fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chemistry is suitable for in vitro labeling experiments, and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.

Monofunctional Carbocyanine Dyes for Bio- and Bioorthogonal Conjugation

Bioconjugate Chemistry. Dec, 2008  |  Pubmed ID: 19053316

A facile synthetic route to prepare monofunctional carbocyanine dyes for biological application is developed. Three pentamethine carbocyanine dyes have been successfully modified with a variety of functional groups such as: carboxylic acids, azides, or alkynes. The new dyes are characterized by strong NIR fluorescence emission, high extinction coefficients and good quantum yields. The azide and alkyne dyes have potential utility as components in bioorthogonal labeling schemes via [2 + 3] dipolar cycloaddition "click" reactions. The application of one derivative, CyAM-5 alkyne, for bioorthogonal labeling is demonstrated. Fluorescence microscopy shows coupling of CyAM-5 alkyne to Chinese hamster ovary (CHO) cells preincubated with azide modified glycans.

BRAF Activation Initiates but Does Not Maintain Invasive Prostate Adenocarcinoma

PloS One. 2008  |  Pubmed ID: 19079609

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.

Design and Demonstration of a Small-animal Up-conversion Imager

Optics Express. Dec, 2008  |  Pubmed ID: 19104605

This first small-animal up-conversion imager (SAUCI) was developed and used for in-vivo imaging of up-converting nanoparticles (UCNs.) Unlike traditional fluorophores, UCNs absorb multiple lower-energy photons and emit a single higher-energy photon. This unique physical process makes it possible to image deeper into tissue with lower background signals. In vivo imaging of particle accumulation in the liver was demonstrated following intravenous injection of particles.

Improved Detection of Ovarian Cancer Metastases by Intraoperative Quantitative Fluorescence Protease Imaging in a Pre-clinical Model

Gynecologic Oncology. Mar, 2009  |  Pubmed ID: 19135233

Cytoreductive surgery is a cornerstone of therapy in metastatic ovarian cancer. While conventional white light (WL) inspection detects many obvious tumor foci, careful histologic comparison has shown considerable miss rates for smaller foci. The goal of this study was to compare tumor detection using WL versus near infrared (NIR) imaging with a protease activatable probe, as well as to evaluate the ability to quantify NIR fluorescence using a novel quantitative optical imaging system.

18F Labeled Nanoparticles for in Vivo PET-CT Imaging

Bioconjugate Chemistry. Feb, 2009  |  Pubmed ID: 19138113

We report the synthesis and in vivo characterization of an 18F modified trimodal nanoparticle (18F-CLIO). This particle consists of cross-linked dextran held together in core-shell formation by a superparamagnetic iron oxide core and functionalized with the radionuclide 18F in high yield via "click" chemistry. The particle can be detected with positron emission tomography, fluorescence molecular tomography, and magnetic resonance imaging. The presence of 18F dramatically lowers the detection threshold of the nanoparticles, while the facile conjugation chemistry provides a simple platform for rapid and efficient nanoparticle labeling.

Combined Magnetic Resonance and Fluorescence Imaging of the Living Mouse Brain Reveals Glioma Response to Chemotherapy

NeuroImage. Apr, 2009  |  Pubmed ID: 19154791

Fluorescent molecular tomographic (FMT) imaging can noninvasively monitor molecular function in living animals using specific fluorescent probes. However, macroscopic imaging methods such as FMT generally exhibit low anatomical details. To overcome this, we report a quantitative technique to image both structure and function by combining FMT and magnetic resonance (MR) imaging. We show that FMT-MR imaging can produce three-dimensional, multimodal images of living mouse brains allowing for serial monitoring of tumor morphology and protease activity. Combined FMT-MR tumor imaging provides a unique in vivo diagnostic parameter, protease activity concentration (PAC), which reflects histological changes in tumors and is significantly altered by systemic chemotherapy. Alterations in this diagnostic parameter are detectable early after chemotherapy and correlate with subsequent tumor growth, predicting tumor response to chemotherapy. Our results reveal that combined FMT-MR imaging of fluorescent molecular probes could be valuable for brain tumor drug development and other neurological and somatic imaging applications.

Normalized Born Ratio for Fluorescence Optical Projection Tomography

Optics Letters. Feb, 2009  |  Pubmed ID: 19183644

We present a normalized Born approach for fluorescence optical projection tomography that takes into account tissue absorption properties. This approach can be particularly useful to study fluorochrome distribution within tissue. We use the algorithm to three-dimensionally reconstruct and characterize a fluorescein isothiocyanate containing absorptive phantom and an infarcted mouse heart previously injected with a fluorescent molecular probe.

Oncogenic EGFR Signaling Cooperates with Loss of Tumor Suppressor Gene Functions in Gliomagenesis

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2009  |  Pubmed ID: 19196966

Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFR(vIII) do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.

Mutant Sodium Channel for Tumor Therapy

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2009  |  Pubmed ID: 19259066

Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein-Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant.

Assessment of Therapeutic Efficacy and Fate of Engineered Human Mesenchymal Stem Cells for Cancer Therapy

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19264968

The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their "stem-like" properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.

Arterial and Aortic Valve Calcification Abolished by Elastolytic Cathepsin S Deficiency in Chronic Renal Disease

Circulation. Apr, 2009  |  Pubmed ID: 19307473

Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy.

Nanoparticle-target Interactions Parallel Antibody-protein Interactions

Analytical Chemistry. May, 2009  |  Pubmed ID: 19323458

Magnetic particles can act as magnetic relaxation switches (MRSw's) when they bind to target analytes, and switch between their dispersed and aggregated states resulting in changes in the spin-spin relaxation time (T(2)) of their surrounding water protons. Both nanoparticles (NPs, 10-100 nm) and micrometer-sized particles (MPs) have been employed as MRSw's, to sense drugs, metabolites, oligonucleotides, proteins, bacteria, and mammalian cells. To better understand how NPs or MPs interact with targets, we employed as a molecular recognition system the reaction between the Tag peptide of the influenza virus hemagglutinin and a monoclonal antibody to that peptide (anti-Tag). To obtain targets of different size and valency, we attached the Tag peptide to BSA (M(w)= 65000 Daltons, diameter = 8 nm) and to Latex spheres (diameter = 900 nm). To obtain magnetic probes of very different sizes, anti-Tag was conjugated to 40 nm NPs and 1 microm MPs. MP and NP probes reacted with Tag peptide targets in a manner similar to antibody/antigen reactions in solution, exhibiting so-called Prozone effects. MPs detected all types of targets with higher sensitivity than NPs with targets of higher valency being better detected than those of lower valency. The Tag/anti Tag recognition system can be used to synthesize combinations of molecular targets and magnetic probes, to more fully understand the aggregation reaction that occurs when probes bind targets in solution and the ensuing changes in water relaxation times that result.

Molecular Imaging of Innate Immune Cell Function in Transplant Rejection

Circulation. Apr, 2009  |  Pubmed ID: 19332470

Clinical detection of transplant rejection by repeated endomyocardial biopsy requires catheterization and entails risks. Recently developed molecular and cellular imaging techniques that visualize macrophage host responses could provide a noninvasive alternative. Yet, which macrophage functions may provide useful markers for detecting parenchymal rejection remains uncertain.

Optical and Multimodality Molecular Imaging: Insights into Atherosclerosis

Arteriosclerosis, Thrombosis, and Vascular Biology. Jul, 2009  |  Pubmed ID: 19359659

Imaging approaches that visualize molecular targets rather than anatomic structures aim to illuminate vital molecular and cellular aspects of atherosclerosis biology in vivo. Several such molecular imaging strategies stand ready for rapid clinical application. This review describes the growing role of in vivo optical molecular imaging in atherosclerosis and highlights its ability to visualize atheroma inflammation, calcification, and angiogenesis. In addition, we discuss advances in multimodality probes, both in the context of multimodal imaging as well as multifunctional, or "theranostic," nanoparticles. This review highlights particular molecular imaging strategies that possess strong potential for clinical translation.

Heterogeneous in Vivo Behavior of Monocyte Subsets in Atherosclerosis

Arteriosclerosis, Thrombosis, and Vascular Biology. Oct, 2009  |  Pubmed ID: 19372462

Monocytes and macrophages play active roles in atherosclerosis, a chronic inflammatory disease that is a leading cause of death in the developed world. The prevailing paradigm states that, during human atherogenesis, monocytes accumulate in the arterial intima and differentiate into macrophages, which then ingest oxidized lipoproteins, secrete a diverse array of proinflammatory mediators, and eventually become foam cells, the key constituents of a vulnerable plaque. Yet monocytes are heterogeneous. In the mouse, one subset (Ly-6C(hi)) promotes inflammation, expands in hypercholesterolemic conditions, and selectively gives rise to macrophages in atheromata. A different subset (Ly-6C(lo)) attenuates inflammation and promotes angiogenesis and granulation tissue formation in models of tissue injury, but its role in atherosclerosis is largely unknown. In the human, monocyte heterogeneity is preserved but it is still unresolved how subsets correspond functionally. The contradistinctive properties of these cells suggest commitment for specific function before infiltrating tissue. Such commitment argues for discriminate targeting of deleterious subsets while sparing host defense and repair mechanisms. In addition to advancing our understanding of atherosclerosis, the ability to target and image monocyte subsets would allow us to evaluate drugs designed to selectively inhibit monocyte subset recruitment or function, and to stratify patients at risk for developing complications such as myocardial infarction or stroke. In this review we summarize recent advances of our understanding of the behavioral heterogeneity of monocytes during disease progression and outline emerging molecular imaging approaches to address key questions in the field.

Genetically Engineered T Cells to Target EGFRvIII Expressing Glioblastoma

Journal of Neuro-oncology. Sep, 2009  |  Pubmed ID: 19387557

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

Behavior of Endogenous Tumor-associated Macrophages Assessed in Vivo Using a Functionalized Nanoparticle

Neoplasia (New York, N.Y.). May, 2009  |  Pubmed ID: 19412430

Tumor-associated macrophages (TAMs) invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma). AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an "M2" macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

Implantable Diagnostic Device for Cancer Monitoring

Biosensors & Bioelectronics. Jul, 2009  |  Pubmed ID: 19442510

Biopsies provide required information to diagnose cancer but, because of their invasiveness, they are difficult to use for managing cancer therapy. The ability to repeatedly sample the local environment for tumor biomarker, chemotherapeutic agent, and tumor metabolite concentrations could improve early detection of metastasis and personalized therapy. Here we describe an implantable diagnostic device that senses the local in vivo environment. This device, which could be left behind during biopsy, uses a semi-permeable membrane to contain nanoparticle magnetic relaxation switches. A cell line secreting a model cancer biomarker produced ectopic tumors in mice. The transverse relaxation time (T(2)) of devices in tumor-bearing mice was 20+/-10% lower than devices in control mice after 1 day by magnetic resonance imaging (p<0.01). Short term applications for this device are numerous, including verification of successful tumor resection. This may represent the first continuous monitoring device for soluble cancer biomarkers in vivo.

Multimodal Nanoagents for the Detection of Intravascular Thrombi

Bioconjugate Chemistry. Jun, 2009  |  Pubmed ID: 19456115

Thrombosis underlies numerous life-threatening cardiovascular syndromes. Development of thrombosis-specific molecular imaging agents to detect and monitor thrombogenesis and fibrinolysis in vivo could improve the diagnosis, risk stratification, and treatment of thrombosis syndromes. To this end, we have synthesized efficient multimodal nanoagents targeted to two different constituents of thrombi, namely, fibrin and activated factor XIII. These agents are targeted via the conjugation of peptide-targeting ligands to the surface of fluorescently labeled magnetic nanoparticles. As demonstrated by in vitro and in vivo studies, both nanoagents possess high affinities for thrombi, and enable mutimodal fluorescence and magnetic resonance imaging.

A Multifunctional Single-attachment-point Reagent for Controlled Protein Biotinylation

Bioconjugate Chemistry. Jan, 2009  |  Pubmed ID: 19072220

The biotin/avidin system is one of the most widely used affinity detection and affinity capture systems in biology. However, the determination of the exact number of biotin tags attached onto a substrate is complicated by the fact that biotin does not present any light-absorbing or -emitting properties. Here, we describe a fluorescent biotinylation reagent designed from the general multifunctional single-attachment-point (MSAP) reagent concept. A Lys-Cys dipeptide scaffold was used to display a biotin functional group and a fluorescein functional group along with an N-hydroxysuccinimide ester reactive group. The resulting bifunctional MSAP reagent, Fl-Biotin-NHS, was used to prepare a monobiotinylated version of cetuximab, which was further reacted with avidin to obtain a soluble avidin-based cetuximab oligomer. The MSAP peptide-scaffold approach allows fluorophores, chromophores, or reactive groups to be combined with biotin and provides a broad approach to obtain multifunctional biotin-based reagents.

Enhancing Navigation in Biomedical Databases by Community Voting and Database-driven Text Classification

BMC Bioinformatics. 2009  |  Pubmed ID: 19799796

The breadth of biological databases and their information content continues to increase exponentially. Unfortunately, our ability to query such sources is still often suboptimal. Here, we introduce and apply community voting, database-driven text classification, and visual aids as a means to incorporate distributed expert knowledge, to automatically classify database entries and to efficiently retrieve them.

Oxazine Conjugated Nanoparticle Detects in Vivo Hypochlorous Acid and Peroxynitrite Generation

Journal of the American Chemical Society. Nov, 2009  |  Pubmed ID: 19817443

The current lack of suitable probes has limited the in vivo imaging of reactive oxygen/nitrogen species (ROS/RNS). ROS/RNS are often generated by ischemia-induced inflammation; defining the extent of tissue involvement or ROS/RNS-related damage would have a significant clinical impact. We present the preparation and demonstration of a fluorogenic sensor for monitoring peroxynitrite (ONOO(-)) and myeloperoxidase (MPO) mediated hypochlorous acid (HOCl/OCl(-)) production. The sensor consists of a long circulating biocompatible nanoparticle that targets phagocytic cells in vivo and is coated with approximately 400 quenched oxazine fluorophores that are released by reaction with HOCl or ONOO(-) but are stable toward oxidants such as hydroxyl radical, hydrogen peroxide, and superoxide. MPO-dependent probe activation is chloride ion dependent and is negated in flow cytometry studies of MPO inhibitor treated neutrophils. Fluorescence reflectance imaging and microscopic fluorescence imaging in mouse hearts after myocardial infarction showed probe release into neutrophil-rich ischemic areas, making this ROS/RNS sensor a novel prognostic indicator.

18F-4V for PET-CT Imaging of VCAM-1 Expression in Atherosclerosis

JACC. Cardiovascular Imaging. Oct, 2009  |  Pubmed ID: 19833312

The aim of this study was to iteratively develop and validate an (18)F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography-computed tomography (PET-CT) in murine atherosclerotic arteries.

Ultrasensitive Detection of Bacteria Using Core-shell Nanoparticles and an NMR-filter System

Angewandte Chemie (International Ed. in English). 2009  |  Pubmed ID: 19554581

Upconverting Luminescent Nanomaterials: Application to in Vivo Bioimaging

Chemical Communications (Cambridge, England). Jul, 2009  |  Pubmed ID: 19585016

In this report, the development of multi-channel anti-Stokes luminescent Y2O3 nanoparticles for application to in vivo upconversion imaging is detailed.

Hybrid in Vivo FMT-CT Imaging of Protease Activity in Atherosclerosis with Customized Nanosensors

Arteriosclerosis, Thrombosis, and Vascular Biology. Oct, 2009  |  Pubmed ID: 19608968

Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT).

Monocyte Subset Dynamics in Human Atherosclerosis Can Be Profiled with Magnetic Nano-sensors

PloS One. 2009  |  Pubmed ID: 19461894

Monocytes are circulating macrophage and dendritic cell precursors that populate healthy and diseased tissue. In humans, monocytes consist of at least two subsets whose proportions in the blood fluctuate in response to coronary artery disease, sepsis, and viral infection. Animal studies have shown that specific shifts in the monocyte subset repertoire either exacerbate or attenuate disease, suggesting a role for monocyte subsets as biomarkers and therapeutic targets. Assays are therefore needed that can selectively and rapidly enumerate monocytes and their subsets. This study shows that two major human monocyte subsets express similar levels of the receptor for macrophage colony stimulating factor (MCSFR) but differ in their phagocytic capacity. We exploit these properties and custom-engineer magnetic nanoparticles for ex vivo sensing of monocytes and their subsets. We present a two-dimensional enumerative mathematical model that simultaneously reports number and proportion of monocyte subsets in a small volume of human blood. Using a recently described diagnostic magnetic resonance (DMR) chip with 1 microl sample size and high throughput capabilities, we then show that application of the model accurately quantifies subset fluctuations that occur in patients with atherosclerosis.

The Antiproliferative Cytostatic Effects of a Self-activating Viridin Prodrug

Molecular Cancer Therapeutics. Jun, 2009  |  Pubmed ID: 19509266

Although viridins like wortmannin (Wm) have long been examined as anticancer agents, their ability to self-activate has only recently been recognized. Here, we describe the cytostatic effects of a self-activating viridin (SAV), which is an inactive, polymeric prodrug. SAV self-activates to generate a bioactive, fluorescent viridin NBD-Wm with a half-time of 9.2 hours. With cultured A549 cells, 10 micromol/L SAV caused growth arrest without inducing apoptosis or cell death, a cytostatic action markedly different from other chemotherapeutic agents (vinblastine, camptothecin, and paclitaxel). In vivo, a SAV dosing of 1 mg/kg once in 48 hours (i.p.) resulted in growth arrest of an A549 tumor xenograft, with growth resuming when dosing ceased. With a peak serum concentration of SAV of 2.36 micromol/L (at 2 hours post i.p. injection), the concentration of bioactive NBD-Wm was 41 nmol/L based on the partial inhibition of neutrophil respiratory burst. Therefore, SAV was present as an inactive prodrug in serum (peak = 2.36 micromol/L), which generated low concentrations of active viridin (41 nmol/L). SAV is a prodrug, the slow release and cytostatic activities of which suggest that it might be useful as a component of metronomic-based chemotherapeutic strategies.

A Stabilized Demethoxyviridin Derivative Inhibits PI3 Kinase

Bioorganic & Medicinal Chemistry Letters. Aug, 2009  |  Pubmed ID: 19523825

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26min versus Wm's half-life of 3470min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1min. To overcome Dmv's instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218min in PBS and 64min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC(50) of 44nM, compared to Wm's IC(50) of 12nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems.

On the Dual Contrast Enhancement Mechanism in Frequency-selective Inversion-recovery Magnetic Resonance Angiography (IRON-MRA)

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Aug, 2009  |  Pubmed ID: 19526511

The susceptibility of blood changes after administration of a paramagnetic contrast agent that shortens T(1). Concomitantly, the resonance frequency of the blood vessels shifts in a geometry-dependent way. This frequency change may be exploited for incremental contrast generation by applying a frequency-selective saturation prepulse prior to the imaging sequence. The dual origin of vascular enhancement depending first on off-resonance and second on T(1) lowering was investigated in vitro, together with the geometry dependence of the signal at 3T. First results obtained in an in vivo rabbit model are presented.

Efficacy of Sunitinib and Radiotherapy in Genetically Engineered Mouse Model of Soft-tissue Sarcoma

International Journal of Radiation Oncology, Biology, Physics. Jul, 2009  |  Pubmed ID: 19545786

Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS).

Lymphotropic Nanoparticle-enhanced Magnetic Resonance Imaging (LNMRI) Identifies Occult Lymph Node Metastases in Prostate Cancer Patients Prior to Salvage Radiation Therapy

Clinical Imaging. Jul-Aug, 2009  |  Pubmed ID: 19559353

Twenty-six patients with prostate cancer status post-radical prostatectomy who were candidates for salvage radiation therapy (SRT) underwent lymphotropic nanoparticle enhanced MRI (LNMRI) using superparamagnetic nanoparticle ferumoxtran-10. LNMRI was well tolerated, with only two adverse events, both Grade 2. Six (23%) of the 26 patients, previously believed to be node negative, tested lymph node positive by LNMRI. A total of nine positive lymph nodes were identified in these six patients, none of which were enlarged based on size criteria.

Nuclear Microscopy: a Novel Technique for Quantitative Imaging of Gadolinium Distribution Within Tissue Sections

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada. Aug, 2009  |  Pubmed ID: 19575834

All clinically-approved and many novel gadolinium (Gd)-based contrast agents used to enhance signal intensity in magnetic resonance imaging (MRI) are optically silent. To verify MRI results, a "gold standard" that can map and quantify Gd down to the parts per million (ppm) levels is required. Nuclear microscopy is a relatively new technique that has this capability and is composed of a combination of three ion beam techniques: scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle induced X-ray emission used in conjunction with a high energy proton microprobe. In this proof-of-concept study, we show that in diseased aortic vessel walls obtained at 2 and 4 h after intravenous injection of the myeloperoxidase-sensitive MRI agent, bis-5-hydroxytryptamide-diethylenetriamine-pentaacetate gadolinium, there was a time-dependant Gd clearance (2 h = 18.86 ppm, 4 h = 8.65 ppm). As expected, the control animal, injected with the clinically-approved conventional agent diethylenetriamine-pentaacetate gadolinium and sacrificed 1 week after injection, revealed no significant residual Gd in the tissue. Similar to known in vivo Gd pharmacokinetics, we found that Gd concentration dropped by a factor of 2 in vessel wall tissue in 1.64 h. Further high-resolution studies revealed that Gd was relatively uniformly distributed, consistent with random agent diffusion. We conclude that nuclear microscopy is potentially very useful for validation studies involving Gd-based magnetic resonance contrast agents.

A DNA-binding Gd Chelate for the Detection of Cell Death by MRI

Chemical Communications (Cambridge, England). Aug, 2009  |  Pubmed ID: 19597620

GadoTO, a MR contrast agent for the detection of cell death, consists of a nucleic acid-binding fluorophore attached to a gadolinium chelate.

Synthesis and Photophysical Properties of Sulfonamidophenyl Porphyrins As Models for Activatable Photosensitizers

The Journal of Organic Chemistry. Aug, 2009  |  Pubmed ID: 19610602

The ability to localize agents to specific anatomic sites remains an important aspect in designing more efficient therapeutics. Light-activated therapies, in particular, allow for the focal ablation of target tissues and cells. In order to increase the specificity of these agents, stimuli-activated systems have been developed, which are nonphototoxic in the absence of activation. To this end, we propose a novel paradigm for excited state quenching and activation based upon the direct conjugation of quenching moieties to the porphyrinic macrocycle. Model compounds, based upon meso-(p-aminophenyl)porphyrins were synthesized bearing 1 to 4 sulfonamide-linked 2,4-dinitrobenzene. The singlet oxygen and fluorescence quantum yields of these compounds were obtained and compared, as well as the kinetics of activation with relevant activating agents. In addition, methods were developed to further modify the porphyrin in order to modulate the polarity and effect conjugation to biomolecules or nanoparticulate scaffolds. These systems may prove useful in the treatment of a number of disease states, such as cancer and bacterial infection.

Rapid Detection and Profiling of Cancer Cells in Fine-needle Aspirates

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2009  |  Pubmed ID: 19620715

There is a growing need for fast, highly sensitive and quantitative technologies to detect and profile unaltered cells in biological samples. Technologies in current clinical use are often time consuming, expensive, or require considerable sample sizes. Here, we report a diagnostic magnetic resonance (DMR) sensor that combines a miniaturized NMR probe with targeted magnetic nanoparticles for detection and molecular profiling of cancer cells. The sensor measures the transverse relaxation rate of water molecules in biological samples in which target cells of interest are labeled with magnetic nanoparticles. We achieved remarkable sensitivity improvements over our prior DMR prototypes by synthesizing new nanoparticles with higher transverse relaxivity and by optimizing assay protocols. We detected as few as 2 cancer cells in 1-microL sample volumes of unprocessed fine-needle aspirates of tumors and profiled the expression of several cellular markers in <15 min.

Identification of Splenic Reservoir Monocytes and Their Deployment to Inflammatory Sites

Science (New York, N.Y.). Jul, 2009  |  Pubmed ID: 19644120

A current paradigm states that monocytes circulate freely and patrol blood vessels but differentiate irreversibly into dendritic cells (DCs) or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes assemble in clusters in the cords of the subcapsular red pulp and are distinct from macrophages and DCs. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue, and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes and identify splenic monocytes as a resource that the body exploits to regulate inflammation.

Abrogation of Antibody-induced Arthritis in Mice by a Self-activating Viridin Prodrug and Association with Impaired Neutrophil and Endothelial Cell Function

Arthritis and Rheumatism. Aug, 2009  |  Pubmed ID: 19644878

To test a novel self-activating viridin (SAV) prodrug that slowly releases wortmannin, a potent phosphoinositide 3-kinase inhibitor, in a model of antibody-mediated inflammatory arthritis.

Enzyme-sensitive Magnetic Resonance Imaging Targeting Myeloperoxidase Identifies Active Inflammation in Experimental Rabbit Atherosclerotic Plaques

Circulation. Aug, 2009  |  Pubmed ID: 19652086

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.

High-yielding Syntheses of Hydrophilic Conjugatable Chlorins and Bacteriochlorins

Organic & Biomolecular Chemistry. Sep, 2009  |  Pubmed ID: 19675897

Next-generation photodynamic therapy agents based upon the conjugation of multiple photosensitizers to a targeting backbone will allow for more efficacious light-based therapies. To this end, we have developed glucose-modified chlorins and bacteriochlorins featuring a reactive carboxylic acid linker for conjugation to targeting moieties. The photosensitizers were synthesized in relatively high yields from meso-tetra(p-aminophenyl)porphyrin, and resulted in neutral, hydrophilic chromophores with superb absorption profiles in the far-red and near-infrared portions of the electromagnetic spectrum. In addition, conjugation of these photosensitizers to a model nanoscaffold (crosslinked dextran-coated nanoparticles) demonstrated that the inclusion of hydrophilic sugar moieties increased the number of dyes that can be loaded while maintaining suspension stability. The described compounds are expected to be particularly useful in the synthesis of a number of targeted nanotherapeutic systems.

Fast and Sensitive Pretargeted Labeling of Cancer Cells Through a Tetrazine/trans-cyclooctene Cycloaddition

Angewandte Chemie (International Ed. in English). 2009  |  Pubmed ID: 19697389

MRI of a Novel Murine Working Heart Transplant Model

Circulation. Heart Failure. May, 2009  |  Pubmed ID: 19718275

Transillumination Fluorescence Imaging in Mice Using Biocompatible Upconverting Nanoparticles

Optics Letters. Sep, 2009  |  Pubmed ID: 19724491

We report on a systematic study of upconverting fluorescence signal generation within turbid phantoms and real tissues. An accurate three-point Green's function, describing the forward model of photon propagation, is established and experimentally validated. We further demonstrate, for the first time to our knowledge, autofluorescence-free transillumination imaging of mice that have received biocompatible upconverting nanoparticles. The method holds great promise for artifact-free whole-body visualization of optical molecular probes.

Non-invasive Optical Detection of Cathepsin K-mediated Fluorescence Reveals Osteoclast Activity in Vitro and in Vivo

Bone. Feb, 2009  |  Pubmed ID: 19007918

Osteoclasts degrade bone matrix by demineralization followed by degradation of type I collagen through secretion of the cysteine protease, cathepsin K. Current imaging modalities are insufficient for sensitive observation of osteoclast activity, and in vivo live imaging of osteoclast resorption of bone has yet to be demonstrated. Here, we describe a near-infrared fluorescence reporter probe whose activation by cathepsin K is shown in live osteoclast cells and in mouse models of development and osteoclast upregulation. Cathepsin K probe activity was monitored in live osteoclast cultures and correlates with cathepsin K gene expression. In ovariectomized mice, cathepsin K probe upregulation precedes detection of bone loss by micro-computed tomography. These results are the first to demonstrate non-invasive visualization of bone degrading enzymes in models of accelerated bone loss, and may provide a means for early diagnosis of upregulated resorption and rapid feedback on efficacy of treatment protocols prior to significant loss of bone in the patient.

High Throughput Transmission Optical Projection Tomography Using Low Cost Graphics Processing Unit

Optics Express. Dec, 2009  |  Pubmed ID: 20052155

We implement the use of a graphics processing unit (GPU) in order to achieve real time data processing for high-throughput transmission optical projection tomography imaging. By implementing the GPU we have obtained a 300 fold performance enhancement in comparison to a CPU workstation implementation. This enables to obtain on-the-fly reconstructions enabling for high throughput imaging.

A Wortmannin-cetuximab As a Double Drug

Bioconjugate Chemistry. Nov, 2009  |  Pubmed ID: 19883074

Double drugs are obtained when two pharmacologically active entities are covalently joined to improve potency. We conjugated the viridin Wm with a self-activating linkage to cetuximab and demonstrated the retention of immunoreactivity by the conjugate. Though cetuximab lacked a growth inhibitory activity against A549 cells, the Wm-cetuximab conjugate had an antiproliferative IC(50) of 155 nM in vitro. The chemistry of attaching a self-releasing Wm to clinically approved antibodies is general and, in selected instances, may yield antibody-based double drugs with improved efficacy.

Molecular MRI of Cardiomyocyte Apoptosis with Simultaneous Delayed-enhancement MRI Distinguishes Apoptotic and Necrotic Myocytes in Vivo: Potential for Midmyocardial Salvage in Acute Ischemia

Circulation. Cardiovascular Imaging. Nov, 2009  |  Pubmed ID: 19920044

A novel dual-contrast molecular MRI technique to image both cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of ischemia is presented. The technique uses the annexin-based nanoparticle AnxCLIO-Cy5.5 (apoptosis) and simultaneous delayed-enhancement imaging with a novel gadolinium chelate, Gd-DTPA-NBD (necrosis).

Molecular MRI Detects Low Levels of Cardiomyocyte Apoptosis in a Transgenic Model of Chronic Heart Failure

Circulation. Cardiovascular Imaging. Nov, 2009  |  Pubmed ID: 19920045

The ability to image cardiomyocyte (CM) apoptosis in heart failure could facilitate more accurate diagnostics and optimize targeted therapeutics. We thus aimed to develop a platform to image CM apoptosis quantitatively and specifically in heart failure in vivo. The myocardium in heart failure, however, is characterized by very low levels of CM apoptosis and normal vascular permeability, factors thought to preclude the use of molecular MRI.

Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model

PloS One. 2009  |  Pubmed ID: 19956597

Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time.

Diffractionless Beam in Free Space with Adiabatic Changing Refractive Index in a Single Mode Tapered Slab Waveguide

Optics Express. Nov, 2009  |  Pubmed ID: 19997414

We propose a novel design to produce a free space diffractionless beam by adiabatically reducing the difference of the refractive index between the core and the cladding regions of a single mode tapered slab waveguide. To ensure only one propagating eigenmode in the adiabatic transition, the correlation of the waveguide core width and the refractive index is investigated. Under the adiabatic condition, we demonstrate that our waveguide can emit a diffractionless beam in free space up to 500 micrometers maintaining 72% of its original peak intensity. The proposed waveguide could find excellent applications for imaging purposes where an extended depth of field is required.

Unbiased Discovery of in Vivo Imaging Probes Through in Vitro Profiling of Nanoparticle Libraries

Integrative Biology : Quantitative Biosciences from Nano to Macro. Apr, 2009  |  Pubmed ID: 20023731

In vivo imaging reveals how proteins and cells function as part of complex regulatory networks in intact organisms, and thereby contributes to a systems-level understanding of biological processes. However, the development of novel in vivo imaging probes remains challenging. Most probes are directed against a limited number of pre-specified protein targets; cell-based screens for imaging probes have shown promise, but raise concerns over whether in vitro surrogate cell models recapitulate in vivo phenotypes. Here, we rapidly profile the in vitro binding of nanoparticle imaging probes in multiple samples of defined target vs. background cell types, using primary cell isolates. This approach selects for nanoparticles that show desired targeting effects across all tested members of a class of cells, and decreases the likelihood that an idiosyncratic cell line will unduly skew screening results. To adjust for multiple hypothesis testing, we use permutation methods to identify nanoparticles that best differentiate between the target and background cell classes. (This approach is conceptually analogous to one used for high-dimensionality datasets of genome-wide gene expression, e.g. to identify gene expression signatures that discriminate subclasses of cancer.) We apply this approach to the identification of nanoparticle imaging probes that bind endothelial cells, and validate our in vitro findings in human arterial samples, and by in vivo intravital microscopy in mice. Overall, this work presents a generalizable approach to the unbiased discovery of in vivo imaging probes, and may guide the further development of novel endothelial imaging probes.

Early Identification of Aortic Valve Sclerosis Using Iron Oxide Enhanced MRI

Journal of Magnetic Resonance Imaging : JMRI. Jan, 2010  |  Pubmed ID: 20027578

To test the ability of MION-47 enhanced MRI to identify tissue macrophage infiltration in a rabbit model of aortic valve sclerosis (AVS).

Binding Affinity and Kinetic Analysis of Targeted Small Molecule-modified Nanoparticles

Bioconjugate Chemistry. Jan, 2010  |  Pubmed ID: 20028085

Nanoparticles bearing surface-conjugated targeting ligands are increasingly being explored for a variety of biomedical applications. The multivalent conjugation of targeting ligands on the surface of nanoparticles is presumed to enhance binding to the desired target. However, given the complexities inherent in the interactions of nanoparticle surfaces with proteins, and the structural diversity of nanoparticle scaffolds and targeting ligands, our understanding of how conjugation of targeting ligands affects nanoparticle binding remains incomplete. Here, we use surface plasmon resonance (SPR) to directly and quantitatively study the affinity and binding kinetics of nanoparticles that display small molecules conjugated to their surface. We studied the interaction between a single protein target and a structurally related series of targeting ligands whose intrinsic affinity varies over a 4500-fold range and performed SPR at protein densities that reflect endogenous receptor densities. We report that even weak small molecule targeting ligands can significantly enhance target-specific avidity (by up to 4 orders of magnitude) through multivalent interactions and also observe a much broader range of kinetic effects than has been previously reported. Quantitative measurement of how the affinity and kinetics of nanoparticle binding vary as a function of different surface conjugations is a rapid, generalizable approach to nanoparticle characterization that can inform the design of nanoparticles for biomedical applications.

Intraoperative Near-infrared Fluorescent Cholangiography (NIRFC) in Mouse Models of Bile Duct Injury

World Journal of Surgery. Feb, 2010  |  Pubmed ID: 20033407

Accidental injury to the common bile duct is a rare but serious complication of laparoscopic cholecystectomy. Accurate visualization of the biliary ducts may prevent injury or allow its early detection. Conventional X-ray cholangiography is often used and can mitigate the severity of injury when correctly interpreted. However, it may be useful to have an imaging method that could provide real-time extrahepatic bile duct visualization without changing the field of view from the laparoscope. The purpose of the present study was to test a new near-infrared (NIR) fluorescent agent that is rapidly excreted via the biliary route in preclinical models to evaluate intraoperative real-time near infrared fluorescent cholangiography (NIRFC).

Activatable Magnetic Resonance Imaging Agents for Myeloperoxidase Sensing: Mechanism of Activation, Stability, and Toxicity

Journal of the American Chemical Society. Jan, 2010  |  Pubmed ID: 19968300

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.

Myeloperoxidase-rich Ly-6C+ Myeloid Cells Infiltrate Allografts and Contribute to an Imaging Signature of Organ Rejection in Mice

The Journal of Clinical Investigation. Jul, 2010  |  Pubmed ID: 20577051

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.

Near-infrared Fluorescent Probe for Imaging of Pancreatic Beta Cells

Bioconjugate Chemistry. Jul, 2010  |  Pubmed ID: 20583828

The ability to image and ultimately quantitate beta-cell mass in vivo will likely have far reaching implications in the study of diabetes biology, in the monitoring of disease progression or response to treatment, and for drug development. Here, using animal models, we report on the synthesis, characterization, and intravital microscopic imaging properties of a near-infrared fluorescent exendin-4 analogue with specificity for the GLP-1 receptor on beta cells (E4(K12)-Fl). The agent demonstrated subnanomolar EC(50) binding concentrations, with high specificity and binding that could be inhibited by GLP-1R agonists. Following intravenous administration to mice, pancreatic islets were readily distinguishable from exocrine pancreas, achieving target-to-background ratios within the pancreas of 6:1, as measured by intravital microscopy. Serial imaging revealed rapid accumulation kinetics (with initial signal within the islets detectable within 3 min and peak fluorescence within 20 min of injection), making this an ideal agent for in vivo imaging.

Arterial and Aortic Valve Calcification Inversely Correlates with Osteoporotic Bone Remodelling: a Role for Inflammation

European Heart Journal. Aug, 2010  |  Pubmed ID: 20601388

Westernized countries face a growing burden of cardiovascular calcification and osteoporosis. Despite its vast clinical significance, the precise nature of this reciprocal relationship remains obscure. We hypothesize that cardiovascular calcification progresses with inflammation and inversely correlates with bone tissue mineral density (TMD).

Bioorthogonal Chemistry Amplifies Nanoparticle Binding and Enhances the Sensitivity of Cell Detection

Nature Nanotechnology. Sep, 2010  |  Pubmed ID: 20676091

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity. Motivated by these properties and by current clinical needs, numerous diagnostic and therapeutic nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

Pioglitazone Suppresses Inflammation in Vivo in Murine Carotid Atherosclerosis: Novel Detection by Dual-target Fluorescence Molecular Imaging

Arteriosclerosis, Thrombosis, and Vascular Biology. Oct, 2010  |  Pubmed ID: 20689078

To investigate the effects of pioglitazone (PIO), a peroxisome proliferator-activated receptor γ agonist, on plaque matrix metalloproteinase (MMP) and macrophage (Mac) responses in vivo in a molecular imaging study.

A Light-activated Theranostic Nanoagent for Targeted Macrophage Ablation in Inflammatory Atherosclerosis

Small (Weinheim an Der Bergstrasse, Germany). Sep, 2010  |  Pubmed ID: 20721949

The synthesis and utility of a multimodal theranostic nanoagent based upon magnetofluorescent nanoparticles for the treatment of inflammatory atherosclerosis is described. These particles are modified with near-infrared fluorophores and light-activated therapeutic moieties, which allow for the optical determination of agent localization and phototoxic activation at spectrally distinct wavelengths. The resulting agent is readily taken up by murine macrophages in vitro and is highly phototoxic, with an LD(50) of 430 pM. Intravenous administration results in the localization of the nanoagent within macrophage-rich atherosclerotic lesions that can be imaged by intravital fluorescence microscopy. Irradiation of the atheroma with 650 nm light activates the therapeutic component and results in eradication of inflammatory macrophages, which may induce lesion stabilization. Importantly, these agents display limited skin photosensitivity, are highly efficacious, and provide an integrated imaging and therapeutic nanoplatform for atherosclerosis.

A Novel Molecule Integrating Therapeutic and Diagnostic Activities Reveals Multiple Aspects of Stem Cell-based Therapy

Stem Cells (Dayton, Ohio). Apr, 2010  |  Pubmed ID: 20127797

Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles.

The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis Via Hif1alpha

Cell. Jan, 2010  |  Pubmed ID: 20141841

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Activin A Promotes Multiple Myeloma-induced Osteolysis and is a Promising Target for Myeloma Bone Disease

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20194748

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-beta family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.

Imaging Primary Lung Cancers in Mice to Study Radiation Biology

International Journal of Radiation Oncology, Biology, Physics. Mar, 2010  |  Pubmed ID: 20206017

To image a genetically engineered mouse model of non-small-cell lung cancer with micro-computed tomography (micro-CT) to measure tumor response to radiation therapy.

Bioorthogonal Turn-On Probes for Imaging Small Molecules Inside Living Cells

Angewandte Chemie (International Ed. in English). Apr, 2010  |  Pubmed ID: 20306505

Glowing tags: A series of activatable ("turn-on") tetrazine-conjugated fluorescent probes was developed, which react rapidly in an inverse-electron-demand [4+2] cycloaddition with strained dienophiles such as trans-cyclooctene, thereby strongly increasing the fluorescence intensity. The novel turn-on probes were applied for intracellular live-cell imaging of a microtubuli-binding trans-cyclooctene modified taxol.

Magnetic Nanoparticle Biosensors

Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. May-Jun, 2010  |  Pubmed ID: 20336708

One of the major challenges in medicine is the rapid and accurate measurement of protein biomarkers, cells, and pathogens in biological samples. A number of new diagnostic platforms have recently been developed to measure biomolecules and cells with high sensitivity that could enable early disease detection or provide valuable insights into biology at the systems level. Most biological samples exhibit negligible magnetic susceptibility; therefore, magnetic nanoparticles have been used for diverse applications including biosensing, magnetic separation, and thermal ablation therapy. This review focuses on the use of magnetic nanoparticles for detection of biomolecules and cells based on magnetic resonance effects using a general detection platform termed diagnostic magnetic resonance (DMR). DMR technology encompasses numerous assay configurations and sensing principles, and to date magnetic nanoparticle biosensors have been designed to detect a wide range of targets including DNA/mRNA, proteins, enzymes, drugs, pathogens, and tumor cells. The core principle behind DMR is the use of magnetic nanoparticles as proximity sensors that modulate the spin-spin relaxation time of neighboring water molecules, which can be quantified using clinical MRI scanners or benchtop nuclear magnetic resonance (NMR) relaxometers. Recently, the capabilities of DMR technology were advanced considerably with the development of miniaturized, chip-based NMR (microNMR) detector systems that are capable of performing highly sensitive measurements on microliter sample volumes and in multiplexed format. With these and future advances in mind, DMR biosensor technology holds considerable promise to provide a high-throughput, low-cost, and portable platform for large scale molecular and cellular screening in clinical and point-of-care settings.

Imaging of Molecular Probe Activity with Born-normalized Fluorescence Optical Projection Tomography

Optics Letters. Apr, 2010  |  Pubmed ID: 20364226

Optical projection tomography is a new ex vivo imaging technique that allows imaging of whole organs in three dimensions at high spatial resolutions. In this Letter we demonstrate its capability to tomographically visualize molecular activity in whole organs of mice. In particular, eosinophil activity in asthmatic lungs is resolved using a Born-normalized fluorescence optical projection tomography and employing a near-IR molecular probe. The possibility to achieve molecularly sensitive imaging contrast in optical projection tomography by means of targeted and activatable imaging reporter agents adds a new range of capabilities for investigating molecular signatures of pathophysiological processes and a wide variety of diseases and their development.

Impaired Infarct Healing in Atherosclerotic Mice with Ly-6C(hi) Monocytosis

Journal of the American College of Cardiology. Apr, 2010  |  Pubmed ID: 20378083

The aim of this study was to test whether blood monocytosis in mice with atherosclerosis affects infarct healing.

Hybrid PET-optical Imaging Using Targeted Probes

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2010  |  Pubmed ID: 20385821

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a (18)F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r(2) > 0.99) and spatial signal distribution (r(2) > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.

Rapid Biocompatibility Analysis of Materials Via in Vivo Fluorescence Imaging of Mouse Models

PloS One. 2010  |  Pubmed ID: 20386609

Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis.

The Vascular Biology of Atherosclerosis and Imaging Targets

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. May, 2010  |  Pubmed ID: 20395349

The growing worldwide health challenge of atherosclerosis, together with advances in imaging technologies, have stimulated considerable interest in novel approaches to gauging this disease. The last several decades have witnessed a burgeoning in understanding of the molecular pathways involved in atherogenesis, lesion progression, and the mechanisms underlying the complications of human atherosclerotic plaques. The imaging of atherosclerosis is reaching beyond anatomy to encompass assessment of aspects of plaque biology related to the pathogenesis and complication of the disease. The harnessing of these biologic insights promises to provide a plethora of new targets for molecular imaging of atherosclerosis. The goals for the years to come must include translation of the experimental work to visualization of these appealing biologic targets in humans.

Development of a Bioorthogonal and Highly Efficient Conjugation Method for Quantum Dots Using Tetrazine-norbornene Cycloaddition

Journal of the American Chemical Society. Jun, 2010  |  Pubmed ID: 20481508

We present a bioorthogonal and modular conjugation method for efficient coupling of organic dyes and biomolecules to quantum dots (QDs) using a norbornene-tetrazine cycloaddition. The use of noncoordinating functional groups combined with the rapid rate of the cycloaddition leads to highly efficient conjugation. We have applied this method to the in situ targeting of norbornene-coated QDs to live cancer cells labeled with tetrazine-modified proteins.

The Phosphoinositide 3-kinase Regulatory Subunit P85alpha Can Exert Tumor Suppressor Properties Through Negative Regulation of Growth Factor Signaling

Cancer Research. Jul, 2010  |  Pubmed ID: 20530665

Phosphoinositide 3-kinase (PI3K) plays a critical role in tumorigenesis, and the PI3K p85 regulatory subunit exerts both positive and negative effects on signaling. Expression of Pik3r1, the gene encoding p85, is decreased in human prostate, lung, ovarian, bladder, and liver cancers, consistent with the possibility that p85 has tumor suppressor properties. We tested this hypothesis by studying mice with a liver-specific deletion of the Pik3r1 gene. These mice exhibited enhanced insulin and growth factor signaling and progressive changes in hepatic pathology, leading to the development of aggressive hepatocellular carcinomas with pulmonary metastases. Liver tumors that arose exhibited markedly elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, along with Akt activation and decreased PTEN expression, at both the mRNA and protein levels. Together, these results substantiate the concept that the p85 subunit of PI3K has a tumor-suppressive role in the liver and possibly other tissues.

Molecular Imaging of Atherosclerosis: a Progress Report

Texas Heart Institute Journal / from the Texas Heart Institute of St. Luke's Episcopal Hospital, Texas Children's Hospital. 2010  |  Pubmed ID: 20548813

Intravital Molecular Imaging of Small-diameter Tissue-engineered Vascular Grafts in Mice: a Feasibility Study

Tissue Engineering. Part C, Methods. Aug, 2010  |  Pubmed ID: 19751103

Creating functional small-diameter tissue-engineered blood vessels has not been successful to date. Moreover, the processes underlying the in vivo remodeling of these grafts and the fate of cells seeded onto scaffolds remain unclear. Here we addressed these unmet scientific needs by using intravital molecular imaging to monitor the development of tissue-engineered vascular grafts (TEVG) implanted in mouse carotid artery.

Magnetic Nanoparticles for Biomedical NMR-based Diagnostics

Beilstein Journal of Nanotechnology. 2010  |  Pubmed ID: 21977404

Rapid and accurate measurements of protein biomarkers, pathogens and cells in biological samples could provide useful information for early disease diagnosis, treatment monitoring, and design of personalized medicine. In general, biological samples have only negligible magnetic susceptibility. Thus, using magnetic nanoparticles for biosensing not only enhances sensitivity but also effectively reduces sample preparation needs. This review focuses on the use of magnetic nanoparticles for in vitro detection of biomolecules and cells based on magnetic resonance effects. This detection platform, termed diagnostic magnetic resonance (DMR), exploits magnetic nanoparticles as proximity sensors, which modulate the spin-spin relaxation time of water molecules surrounding molecularly-targeted nanoparticles. By developing more effective magnetic nanoparticle biosensors, DMR detection limits for various target moieties have been considerably improved over the last few years. Already, a library of magnetic nanoparticles has been developed, in which a wide range of targets, including DNA/mRNA, proteins, small molecules/drugs, bacteria, and tumor cells, have been quantified. More recently, the capabilities of DMR technology have been further advanced with new developments such as miniaturized nuclear magnetic resonance detectors, better magnetic nanoparticles and novel conjugational methods. These developments have enabled parallel and sensitive measurements to be made from small volume samples. Thus, the DMR technology is a highly attractive platform for portable, low-cost, and efficient biomolecular detection within a biomedical setting.

Angiotensin-converting Enzyme Inhibition Prevents the Release of Monocytes from Their Splenic Reservoir in Mice with Myocardial Infarction

Circulation Research. Nov, 2010  |  Pubmed ID: 20930148

Monocytes recruited to ischemic myocardium originate from a reservoir in the spleen, and the release from their splenic niche relies on angiotensin (Ang) II signaling.

High-resolution Magnetic Resonance Imaging Enhanced with Superparamagnetic Nanoparticles Measures Macrophage Burden in Atherosclerosis

Circulation. Oct, 2010  |  Pubmed ID: 20937980

Macrophages contribute to the progression and acute complications of atherosclerosis. Macrophage imaging may serve as a biomarker to identify subclinical inflamed lesions, to predict future risk, and to aid in the assessment of novel therapies.

Enhanced Antitumor Efficacy of Vasculostatin (Vstat120) Expressing Oncolytic HSV-1

Molecular Therapy : the Journal of the American Society of Gene Therapy. Feb, 2010  |  Pubmed ID: 19844198

Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy.

Near-infrared Fluorescence: Application to in Vivo Molecular Imaging

Current Opinion in Chemical Biology. Feb, 2010  |  Pubmed ID: 19879798

Molecular imaging often relies on the use of targeted and activatable reporters to quantitate and visualize targets, biological processes, and cells in vivo. The use of optical probes with near-infrared fluorescence allows for improved photon penetration through tissue and minimizes the effects of tissue autofluorescence. There are several parameters that define the effectiveness of imaging agents in vivo. These factors include probe targeting, activation, pharmacokinetics, biocompatibility, and photophysics. Recent advances in our understanding of these variables as they pertain to the application of optical reporters for in vivo imaging are discussed in this review.

Bioorthogonal Small-molecule Ligands for PARP1 Imaging in Living Cells

Chembiochem : a European Journal of Chemical Biology. Nov, 2010  |  Pubmed ID: 20967817

WNT5A/JNK and FGF/MAPK Pathways Regulate the Cellular Events Shaping the Vertebrate Limb Bud

Current Biology : CB. Nov, 2010  |  Pubmed ID: 21055947

The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown.

Self-assembled Multifunctional Fe/MgO Nanospheres for Magnetic Resonance Imaging and Hyperthermia

Nanomedicine : Nanotechnology, Biology, and Medicine. Apr, 2010  |  Pubmed ID: 19800988

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.

Targeted Nanoagents for the Detection of Cancers

Molecular Oncology. Dec, 2010  |  Pubmed ID: 20851695

Nanotechnology has enabled a renaissance in the diagnosis of cancers. This is due, in part to the ability to develop agents bearing multiple functionalities, including those utilized for targeting, imaging, and therapy, allowing for the tailoring of the properties of the nanomaterials. Whereas many nanomaterials exhibit localization to diseased tissues via intrinsic targeting, the addition of targeting ligands, such as antibodies, peptides, aptamers, and small molecules, facilitates far more sensitive cancer detection. As such, this review focuses upon some of the most poignant examples of the utility of affinity ligand targeted nanoagents in the detection of cancer.

Quantitative Nanostructure-activity Relationship Modeling

ACS Nano. Oct, 2010  |  Pubmed ID: 20857979

Evaluation of biological effects, both desired and undesired, caused by manufactured nanoparticles (MNPs) is of critical importance for nanotechnology. Experimental studies, especially toxicological, are time-consuming, costly, and often impractical, calling for the development of efficient computational approaches capable of predicting biological effects of MNPs. To this end, we have investigated the potential of cheminformatics methods such as quantitative structure-activity relationship (QSAR) modeling to establish statistically significant relationships between measured biological activity profiles of MNPs and their physical, chemical, and geometrical properties, either measured experimentally or computed from the structure of MNPs. To reflect the context of the study, we termed our approach quantitative nanostructure-activity relationship (QNAR) modeling. We have employed two representative sets of MNPs studied recently using in vitro cell-based assays: (i) 51 various MNPs with diverse metal cores (Proc. Natl. Acad. Sci. 2008, 105, 7387-7392) and (ii) 109 MNPs with similar core but diverse surface modifiers (Nat. Biotechnol. 2005, 23, 1418-1423). We have generated QNAR models using machine learning approaches such as support vector machine (SVM)-based classification and k nearest neighbors (kNN)-based regression; their external prediction power was shown to be as high as 73% for classification modeling and having an R(2) of 0.72 for regression modeling. Our results suggest that QNAR models can be employed for: (i) predicting biological activity profiles of novel nanomaterials, and (ii) prioritizing the design and manufacturing of nanomaterials toward better and safer products.

Carboxymethylated Polyvinyl Alcohol Stabilizes Doped Ferrofluids for Biological Applications

Advanced Materials (Deerfield Beach, Fla.). Dec, 2010  |  Pubmed ID: 20859943

In Vivo Imaging in Cancer

Cold Spring Harbor Perspectives in Biology. Dec, 2010  |  Pubmed ID: 20861158

Imaging has become an indispensable tool in the study of cancer biology and in clinical prognosis and treatment. The rapid advances in high resolution fluorescent imaging at single cell level and MR/PET/CT image registration, combined with new molecular probes of cell types and metabolic states, will allow the physical scales imaged by each to be bridged. This holds the promise of translation of basic science insights at the single cell level to clinical application. In this article, we describe the recent advances in imaging at the macro- and micro-scale and how these advances are synergistic with new imaging agents, reporters, and labeling schemes. Examples of new insights derived from the different scales of imaging and relevant probes are discussed in the context of cancer progression and metastasis.

Detection Limits of Intraoperative Near Infrared Imaging for Tumor Resection

Journal of Surgical Oncology. Dec, 2010  |  Pubmed ID: 20872807

The application of fluorescent molecular imaging to surgical oncology is a developing field with the potential to reduce morbidity and mortality. However, the detection thresholds and other requirements for successful intervention remain poorly understood. Here we modeled and experimentally validated depth and size of detection of tumor deposits, trade-offs in coverage and resolution of areas of interest, and required pharmacokinetics of probes based on differing levels of tumor target presentation.

Noninvasive Imaging of Pancreatic Islet Inflammation in Type 1A Diabetes Patients

The Journal of Clinical Investigation. Jan, 2011  |  Pubmed ID: 21123946

Type 1A diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration of the pancreatic islets of Langerhans. A major impediment to advances in understanding, preventing, and curing T1D has been the inability to "see" the disease initiate, progress, or regress, especially during the occult phase. Here, we report the development of a noninvasive method to visualize T1D at the target organ level in patients with active insulitis. Specifically, we visualized islet inflammation, manifest by microvascular changes and monocyte/macrophage recruitment and activation, using magnetic resonance imaging of magnetic nanoparticles (MNPs). As a proof of principle for this approach, imaging of infused ferumoxtran-10 nanoparticles permitted effective visualization of the pancreas and distinction of recent-onset diabetes patients from nondiabetic controls. The observation that MNPs accumulate in the pancreas of T1D patients opens the door to exploiting this noninvasive imaging method to follow T1D progression and monitoring the ability of immunomodulatory agents to clear insulitis.

Dragon (repulsive Guidance Molecule B) Inhibits IL-6 Expression in Macrophages

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2011  |  Pubmed ID: 21187450

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

High-Yielding, Two-Step (18)F Labeling Strategy for (18)F-PARP1 Inhibitors

ChemMedChem. Jan, 2011  |  Pubmed ID: 21207434

Enhanced in Vivo Imaging of Metabolically Biotinylated Cell Surface Reporters

Analytical Chemistry. Feb, 2011  |  Pubmed ID: 21214190

Metabolic biotinylation of intracellular and secreted proteins as well as surface receptors in mammalian cells provides a versatile way to monitor gene expression; to purify and target viral vectors; to monitor cell and tumor distribution in real time in vivo; to label cells for isolation; and to tag proteins for purification, localization, and trafficking. Here, we show that metabolic biotinylation of proteins fused to the bacterial biotin acceptor peptides (BAP) varies among different mammalian cell types and can be enhanced by over 10-fold upon overexpression of the bacterial biotin ligase directed to the same cellular compartment as the fusion protein. We also show that in vivo imaging of metabolically biotinylated cell surface receptors using streptavidin conjugates is significantly enhanced upon coexpression of bacterial biotin ligase in the secretory pathway. These findings have practical applications in designing more efficient targeting and imaging strategies.

Detection of Macrophages in Aortic Aneurysms by Nanoparticle Positron Emission Tomography-computed Tomography

Arteriosclerosis, Thrombosis, and Vascular Biology. Apr, 2011  |  Pubmed ID: 21252070

Current management of aortic aneurysms (AAs) relies primarily on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Because macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 ((18)F) for positron emission tomography-computed tomography (PET-CT) detection of inflammation in AAs.

Micro-NMR for Rapid Molecular Analysis of Human Tumor Samples

Science Translational Medicine. Feb, 2011  |  Pubmed ID: 21346169

Although tumor cells obtained from human patients by image-guided intervention are a valuable source for diagnosing cancer, conventional means of analysis are limited. Here, we report the development of a quantitative micro-NMR (nuclear magnetic resonance) system for rapid, multiplexed analysis of human tumors. We implemented the technology in a clinical setting to analyze cells obtained by fine-needle aspirates from suspected lesions in 50 patients and validated the results in an independent cohort of another 20 patients. Single fine-needle aspirates yielded sufficient numbers of cells to enable quantification of multiple protein markers in all patients within 60 min. Moreover, using a four-protein signature, we report a 96% accuracy for establishing a cancer diagnosis, surpassing conventional clinical analyses by immunohistochemistry. Our results also show that protein expression patterns decay with time, underscoring the need for rapid sampling and diagnosis close to the patient bedside. We also observed a surprising degree of heterogeneity in protein expression both across the different patient samples and even within the same tumor, which has important implications for molecular diagnostics and therapeutic drug targeting. Our quantitative point-of-care micro-NMR technique shows potential for cancer diagnosis in the clinic.

Probing Intracellular Biomarkers and Mediators of Cell Activation Using Nanosensors and Bioorthogonal Chemistry

ACS Nano. Apr, 2011  |  Pubmed ID: 21351804

Nanomaterials offer unique physical properties that make them ideal biosensors for scant cell populations. However, specific targeting of nanoparticles to intracellular proteins has been challenging. Here, we describe a technique to improve intracellular biomarker sensing using nanoparticles that is based on bioorthogonal chemistry. Using trans-cyclooctene-modified affinity ligands that are administered to semipermeabilized cells and revealed by cycloaddition reaction with tetrazine-conjugated nanoparticles, we demonstrate site-specific amplification of nanomaterial binding. We also show that this technique is capable of sensing protein biomarkers and phosho-protein signal mediators, both within the cytosol and nucleus, via magnetic or fluorescent modalities. We expect the described method will have broad applications in nanomaterial-based diagnostics and therapeutics.

In Vivo PET Imaging of Histone Deacetylases by 18F-suberoylanilide Hydroxamic Acid (18F-SAHA)

Journal of Medicinal Chemistry. Aug, 2011  |  Pubmed ID: 21721525

Histone deacetylases (HDACs) are a group of enzymes that modulate gene expression and cell state by deacetylation of both histone and non-histone proteins. A variety of HDAC inhibitors (HDACi) have already undergone clinical testing in cancer. Real-time in vivo imaging of HDACs and their inhibition would be invaluable; however, the development of appropriate imaging agents has remained a major challenge. Here, we describe the development and evaluation of (18)F-suberoylanilide hydroxamic acid ((18)F-SAHA 1a), a close analogue of the most clinically relevant HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). We demonstrate that 1a has near identical biochemical activity profiles to that of SAHA and report findings from pharmacokinetic studies. Using a murine ovarian cancer model, we likewise show that HDAC inhibitor target binding efficacy can be quantitated within 24 h of administration. 1a thus represents the first (18)F-positron emission tomography (PET) HDAC imaging agent, which also exhibits low nanomolar potency and is pharmacologically analogous to a clinically relevant HDAC inhibitor.

Multiplexed Magnetic Labeling Amplification Using Oligonucleotide Hybridization

Advanced Materials (Deerfield Beach, Fla.). Sep, 2011  |  Pubmed ID: 21780311

Synthesis and Evaluation of a Series of 1,2,4,5-tetrazines for Bioorthogonal Conjugation

Bioconjugate Chemistry. Nov, 2011  |  Pubmed ID: 21950520

1,2,4,5-Tetrazines have been established as effective dienes for inverse electron demand [4 + 2] Diels-Alder cycloaddition reactions with strained alkenes for over 50 years. Recently, this reaction pair combination has been applied to bioorthogonal labeling and cell detection applications; however, to date, there has been no detailed examination and optimization of tetrazines for use in biological experiments. Here, we report the synthesis and characterization of 12 conjugatable tetrazines. The tetrazines were all synthesized in a similar fashion and were screened in parallel to identify candidates most ideally suited for biological studies. In depth follow-up studies revealed compounds with varying degrees of stability and reactivity that could each be useful in different bioorthogonal applications. One promising, highly stable, and water-soluble derivative was used in pretargeted cancer cell labeling studies, confirming its utility as a bioorthogonal moiety.

Nanoparticle-mediated Measurement of Target-drug Binding in Cancer Cells

ACS Nano. Nov, 2011  |  Pubmed ID: 21962084

Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells, and drug delivery. The ability to rapidly quantitate drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized NMR technologies, we have developed a magnetic nanoparticle-based approach to directly measure both target expression and drug binding in scant human cells. The method involves covalent conjugation of the small-molecule drug to a magnetic nanoparticle that is then used as a read-out for target expression and drug-binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP in scant cells with excellent correlation to gold standards, the ability to mimic drug pharmacodynamics ex vivo through competitive target-drug binding, and the potential to perform such measurements in clinical samples.

Ubiquitous Detection of Gram-positive Bacteria with Bioorthogonal Magnetofluorescent Nanoparticles

ACS Nano. Nov, 2011  |  Pubmed ID: 21967150

The ability to rapidly diagnose gram-positive pathogenic bacteria would have far reaching biomedical and technological applications. Here we describe the bioorthogonal modification of small molecule antibiotics (vancomycin and daptomycin), which bind to the cell wall of gram-positive bacteria. The bound antibiotics conjugates can be reacted orthogonally with tetrazine-modified nanoparticles, via an almost instantaneous cycloaddition, which subsequently renders the bacteria detectable by optical or magnetic sensing. We show that this approach is specific, selective, fast and biocompatible. Furthermore, it can be adapted to the detection of intracellular pathogens. Importantly, this strategy enables detection of entire classes of bacteria, a feat that is difficult to achieve using current antibody approaches. Compared to covalent nanoparticle conjugates, our bioorthogonal method demonstrated 1-2 orders of magnitude greater sensitivity. This bioorthogonal labeling method could ultimately be applied to a variety of other small molecules with specificity for infectious pathogens, enabling their detection and diagnosis.

Therapeutic SiRNA Silencing in Inflammatory Monocytes in Mice

Nature Biotechnology. Nov, 2011  |  Pubmed ID: 21983520

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

89Zr-labeled Dextran Nanoparticles Allow in Vivo Macrophage Imaging

Bioconjugate Chemistry. Dec, 2011  |  Pubmed ID: 22035047

Tissue macrophages play a critical role both in normal physiology and in disease states. However, because of a lack of specific imaging agents, we continue to have a poor understanding of their absolute numbers, flux rates, and functional states in different tissues. Here, we describe a new macrophage specific positron emission tomography imaging agent, labeled with zirconium-89 ((89)Zr), that was based on a cross-linked, short chain dextran nanoparticle (13 nm). Following systemic administration, the particle demonstrated a vascular half-life of 3.9 h and was found to be located primarily in tissue resident macrophages rather than other white blood cells. Subsequent imaging of the probe using a xenograft mouse model of cancer allowed for quantitation of tumor-associated macrophage numbers, which are of major interest in emerging molecular targeting strategies. It is likely that the material described, which allows the visualization of macrophage biology in vivo, will likewise be useful for a multitude of human applications.

Specific Pathogen Detection Using Bioorthogonal Chemistry and Diagnostic Magnetic Resonance

Bioconjugate Chemistry. Dec, 2011  |  Pubmed ID: 22043803

The development of faster and more sensitive detection methods capable of identifying specific bacterial species and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming microbiological cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified by surface plasmon resonance as well as by highly specific detection of Staphylococcus aureus using a miniaturized diagnostic magnetic resonance system. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction reported here displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings.

Supramolecular Host-Guest Interaction for Labeling and Detection of Cellular Biomarkers

Angewandte Chemie (International Ed. in English). Nov, 2011  |  Pubmed ID: 22113923

Be my guest: A supramolecular host-guest interaction is utilized for highly efficient bioorthogonal labeling of cellular targets. Antibodies labeled with a cyclodextrin host molecule bind to adamantane-labeled magnetofluorescent nanoparticles and provide an amplifiable strategy for biomarker detection that can be adapted to different diagnostic techniques such as molecular profiling or magnetic cell sorting.

Intravital Imaging

Cell. Nov, 2011  |  Pubmed ID: 22118457

Until recently, the idea of observing life deep within the tissues of a living mouse, at a resolution sufficient to pick out cellular behaviors and molecular signals underlying them, remained a much-coveted dream. Now, a new era of intravital fluorescence microscopy has dawned. In this Primer, we review the technologies that made this revolution possible and demonstrate how intravital imaging is beginning to provide quantitative and dynamic insights into cell biology, immunology, tumor biology, and neurobiology.

Molecular Detection of Biomarkers and Cells Using Magnetic Nanoparticles and Diagnostic Magnetic Resonance

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21424441

The rapid and sensitive detection of molecular targets such as proteins, cells, and pathogens in biological specimens is a major focus of ongoing medical research, as it could promote early disease diagnoses and the development of tailored therapeutic strategies. Magnetic nanoparticles (MNP) are attractive candidates for molecular biosensing applications because most biological samples exhibit negligible magnetic susceptibility, and thus the background against which measurements are made is extremely low. Numerous magnetic detection methods exist, but sensing based on magnetic resonance effects has successfully been developed into a general detection platform termed diagnostic magnetic resonance (DMR). DMR technology encompasses numerous assay configurations and sensing principles, and to date magnetic nanoparticle biosensors have been designed to detect a wide range of targets including DNA/mRNA, proteins, enzymes, drugs, pathogens, and tumor cells with exquisite sensitivity. The core principle behind DMR is the use of MNP as proximity sensors that modulate the transverse relaxation time of neighboring water molecules. This signal can be quantified using MR imagers or NMR relaxometers, including miniaturized NMR detector chips that are capable of performing highly sensitive measurements on microliter sample volumes and in a multiplexed format. The speed, sensitivity, and simplicity of the DMR principle, coupled with further advances in NMR biosensor technology should provide a high-throughput, low-cost, and portable platform for large-scale parallel sensing in clinical and point-of-care settings.

Facile Synthesis of Monofunctional Pentamethine Carbocyanine Fluorophores

Dyes and Pigments : an International Journal. Apr, 2011  |  Pubmed ID: 21475610

A high yield route to symmetric, conjugatable pentamethine carbocyanine dyes with far-red/near infrared (NIR) emission between 650 and 700 nm is reported. The dyes are prepared via condensation of indolium or benz[e]indolium inner salts with an alkyl carboxylic acid derivatized malonaldehyde dianil or alternatively in a one-pot reaction without isolation of the malonaldehyde intermediate. The fluorophores are water-soluble, have bright fluorescence emission, are easily prepared in good yield, and are promising candidates for use in a variety of biochemical and in vivo imaging applications.

Indocyanine Green Enables Near-infrared Fluorescence Imaging of Lipid-rich, Inflamed Atherosclerotic Plaques

Science Translational Medicine. May, 2011  |  Pubmed ID: 21613624

New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. Although molecular imaging agents are available for low-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries. Here, we have demonstrated that indocyanine green (ICG), a Food and Drug Administration-approved near-infrared fluorescence (NIRF)-emitting compound, targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aorta, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.

Bioorthogonal Probes for Polo-like Kinase 1 Imaging and Quantification

Angewandte Chemie (International Ed. in English). Sep, 2011  |  Pubmed ID: 21948435

Multicore Assemblies Potentiate Magnetic Properties of Biomagnetic Nanoparticles

Advanced Materials (Deerfield Beach, Fla.). Nov, 2011  |  Pubmed ID: 21953810

Different Capacity of Monocyte Subsets to Phagocytose Iron-oxide Nanoparticles

PloS One. 2011  |  Pubmed ID: 21984904

To explore the capacity of human CD1⁺CD16⁺⁺ and CD14⁺⁺CD16⁻ monocytes to phagocyte iron-oxide nanoparticles in vitro.

Interview: Nanomedicine in Oncology: Miniaturized Means to an Enormous End. Interviewed by Cara Sutton

Nanomedicine (London, England). Nov, 2011  |  Pubmed ID: 22077461

High-yielding, Two-step 18F Labeling Strategy for 18F-PARP1 Inhibitors

ChemMedChem. Mar, 2011  |  Pubmed ID: 21360818

In Vivo Tomographic Imaging of Red-shifted Fluorescent Proteins

Biomedical Optics Express. 2011  |  Pubmed ID: 21483611

We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.

Highly Magnetic Core-shell Nanoparticles with a Unique Magnetization Mechanism

Angewandte Chemie (International Ed. in English). May, 2011  |  Pubmed ID: 21495138

Miniature Magnetic Resonance System for Point-of-care Diagnostics

Lab on a Chip. Jul, 2011  |  Pubmed ID: 21547317

We have developed a next generation, miniaturized platform to diagnose disease at the point-of-care using diagnostic magnetic resonance (DMR-3). Utilizing a rapidly growing library of functionalized magnetic nanoparticles, DMR has previously been demonstrated as a versatile tool to quantitatively and rapidly detect disease biomarkers in unprocessed biological samples. A major hurdle for bringing DMR to the point-of-care has been its sensitivity to temperature variation. As an alternative to costly and bulky mechanisms to control temperature, we have implemented an automated feedback system to track and compensate for the temperature drift, which enables reliable and robust DMR measurements in realistic clinical environments (4-50 °C). Furthermore, the new system interfaces with a mobile device to facilitate system control and data sharing over wireless networks. With such features, the DMR-3 platform can function as a self-contained laboratory even in resource-limited, remote settings. The clinical potential of the new system is demonstrated by detecting trace amounts of proteins and as few as 10 bacteria (Staphylococcus aureus) in a short time frame (<30 min).

Distinguishing Inflammation from Tumor and Peritumoral Edema by Myeloperoxidase Magnetic Resonance Imaging

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jul, 2011  |  Pubmed ID: 21558403

Inflammation occurs routinely when managing gliomas and is not easily distinguishable from tumor regrowth by current MRI methods. The lack of noninvasive technologies that monitor inflammation prevents us to understand whether it is beneficial or detrimental for the patient, and current therapies do not take this host response in consideration. We aim to establish whether a gadolinium (Gd)-based agent targeting the inflammatory enzyme myeloperoxidase (MPO) can selectively detect intra- and peritumoral inflammation as well as glioma response to treatment by MRI. Methods: We carried out serial Gd-bis-5-HT-DTPA (MPO-Gd) MRI before and after treating rodent gliomas with different doses of oncolytic virus (OV) and analyzed animal survival. The imaging results were compared with histopathologic and molecular analyses of the tumors for macrophage/microglia infiltration, virus persistence, and MPO levels.

Report of the National Heart, Lung, and Blood Institute Working Group on the Translation of Cardiovascular Molecular Imaging

Circulation. May, 2011  |  Pubmed ID: 21576680

Biomedical Applications of Tetrazine Cycloadditions

Accounts of Chemical Research. Sep, 2011  |  Pubmed ID: 21627112

Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 10(3) M(-1) s(-1)) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-infrared-emitting fluorophores to dienophile-modifed extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control experiments showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concentrations of labeling agent over a time scale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chemical reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogues, inside living cells. Because the coupling partners are small molecules (<300 Da), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chemistry can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin-avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clinical samples for biomarker profiling to predict malignancy and patient outcome. Finally, we discuss recent results with tetrazine reactions used for in vivo molecular imaging applications. Rapid tetrazine cycloadditions allow modular labeling of small molecules with the most commonly used positron emission tomography isotope, (18)F. Additionally, recent work has applied this reaction directly in vivo for the pretargeted imaging of solid tumors. Future work with tetrazine cycloadditions will undoubtedly lead to optimized protocols, improved probes, and additional biomedical applications.

Dextran-coated Iron Oxide Nanoparticles: a Versatile Platform for Targeted Molecular Imaging, Molecular Diagnostics, and Therapy

Accounts of Chemical Research. Oct, 2011  |  Pubmed ID: 21661727

Advances in our understanding of the genetic basis of disease susceptibility coupled with prominent successes for molecular targeted therapies have resulted in an emerging strategy of personalized medicine. This approach envisions risk stratification and therapeutic selection based on an individual's genetic makeup and physiologic state (the latter assessed through cellular or molecular phenotypes). Molecularly targeted nanoparticles can play a key role in this vision through noninvasive assessments of molecular processes and specific cell populations in vivo, sensitive molecular diagnostics, and targeted delivery of therapeutics. A superparamagnetic iron oxide nanoparticle with a cross-linked dextran coating, or CLIO, is a powerful and illustrative nanoparticle platform for these applications. These structures and their derivatives support diagnostic imaging by magnetic resonance (MRI), optical, and positron emission tomography (PET) modalities and constitute a versatile platform for conjugation to targeting ligands. A variety of conjugation methods exist to couple the dextran surface to different functional groups; in addition, a robust bioorthogonal [4 + 2] cycloaddition reaction between 1,2,4,5-tetrazene (Tz) and trans-cyclooctene (TCO) can conjugate nanoparticles to targeting ligands or label pretargeted cells. The ready availability of conjugation methods has given rise to the synthesis of libraries of small molecule modified nanoparticles, which can then be screened for nanoparticles with specificity for a specific cell type. Since most nanoparticles display their targeting ligands in a multivalent manner, a detailed understanding of the kinetics and affinity of a nanoparticle's interaction with its target (as determined by surface plasmon resonance) can yield functionally important insights into nanoparticle design. In this Account, we review applications of the CLIO platform in several areas relevant to the mission of personalized medicine. We demonstrate rapid and highly sensitive molecular profiling of cancer markers ex vivo, as part of detailed, individualized molecular phenotyping. The CLIO platform also facilitates targeted magnetic resonance and combined modality imaging (such as MR/PET/fluorescence/CT) to enable multiplexed measurement of molecular phenotypes in vivo for early diagnosis and disease classification. Finally, the targeted delivery of a photodynamic therapy agent as part of a theranostic nanoparticle successfully increased local cell toxicity and minimized systemic side effects.

Two-dimensional Intravascular Near-infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-induced Vascular Injury

Journal of the American College of Cardiology. Jun, 2011  |  Pubmed ID: 21679853

This study sought to develop a 2-dimensional (2D) intravascular near-infrared fluorescence (NIRF) imaging strategy for investigation of arterial inflammation in coronary-sized vessels.

Analysis of Mitosis and Antimitotic Drug Responses in Tumors by in Vivo Microscopy and Single-cell Pharmacodynamics

Cancer Research. Jul, 2011  |  Pubmed ID: 21712408

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level.

Accurate Measurement of Pancreatic Islet Beta-cell Mass Using a Second-generation Fluorescent Exendin-4 Analog

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2011  |  Pubmed ID: 21768367

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

In Vivo Detection of Staphylococcus Aureus Endocarditis by Targeting Pathogen-specific Prothrombin Activation

Nature Medicine. Sep, 2011  |  Pubmed ID: 21857652

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Modular Strategy for the Construction of Radiometalated Antibodies for Positron Emission Tomography Based on Inverse Electron Demand Diels-Alder Click Chemistry

Bioconjugate Chemistry. Oct, 2011  |  Pubmed ID: 21877749

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes (64)Cu and (89)Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with (64)Cu and (89)Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7-5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the (64)Cu-DOTA- and (89)Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the (64)Cu- and (89)Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system-one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators-will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.

Human Embryonic Stem Cell-derived Microvascular Grafts for Cardiac Tissue Preservation After Myocardial Infarction

Biomaterials. Feb, 2011  |  Pubmed ID: 21035182

We present use of a synthetic, injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin β4 (Tβ4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + ∼7%, P < 0.001; 6 wk ejection faction, + ∼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue.

A Systems Approach for Tumor Pharmacokinetics

PloS One. 2011  |  Pubmed ID: 21935441

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.

Development of Secreted Protein and Acidic and Rich in Cysteine (SPARC) Targeted Nanoparticles for the Prognostic Molecular Imaging of Metastatic Prostate Cancer

Journal of Nanomedicine & Nanotechnology. Aug, 2011  |  Pubmed ID: 22319675

Prostate cancer is the most commonly diagnosed non-skin malignancy in the United States and presents with a wide range of aggressiveness from extremely slow-growing to highly aggressive. There is a clinical need to determine the metastatic potential of the primary tumor to design the most appropriate treatment plan ranging from watchful waiting to more aggressive, invasive surgical treatments. In this study we have developed a nanoparticle based imaging agent that targets SPARC (Secreted Protein Acidic Rich in Cysteine), a molecular marker of prostate cancer metastatic potential. Previous studies by this group used phage display to identify a peptide with high binding affinity and specificity for SPARC. In this study, the SPARC-targeted peptide sequence was used to design a biomaterial with improved pharmacokinetic properties by attaching it to a biocompatible nanoparticle that is also coupled to a fluorophore for in vivo imaging. Prostate cancer cell lines with varying degrees of SPARC expression were used to show the ability of the targeted nanoparticle to bind specifically to SPARC in vitro and in vivo including the clinically relevant bone and lung metastases. We show that in vivo imaging information correlates with the metastatic potential of the prostate tumor. This prognostic information could enable doctors to stratify patients and design personalized treatment strategies.

Self-assembled Magnetic Filter for Highly Efficient Immunomagnetic Separation

Lab on a Chip. Jan, 2011  |  Pubmed ID: 20949198

We have developed a compact and inexpensive microfluidic chip, the self-assembled magnetic filter, to efficiently remove magnetically tagged cells from suspension. The self-assembled magnetic filter consists of a microfluidic channel built directly above a self-assembled NdFeB magnet. Micrometre-sized grains of NdFeB assemble to form alternating magnetic dipoles, creating a magnetic field with a very strong magnitude B (from the material) and field gradient ▽B (from the configuration) in the microfluidic channel. The magnetic force imparted on magnetic beads is measured to be comparable to state-of-the-art microfabricated magnets, allowing for efficient separations to be performed in a compact, simple device. The efficiency of the magnetic filter is characterized by sorting non-magnetic (polystyrene) beads from magnetic beads (iron oxide). The filter enriches the population of non-magnetic beads to magnetic beads by a factor of >10(5) with a recovery rate of 90% at 1 mL h(-1). The utility of the magnetic filter is demonstrated with a microfluidic device that sorts tumor cells from leukocytes using negative immunomagnetic selection, and concentrates the tumor cells on an integrated membrane filter for optical detection.

Bone Marrow Stromal Cell Transplants Prevent Experimental Enterocolitis and Require Host CD11b+ Splenocytes

Gastroenterology. Mar, 2011  |  Pubmed ID: 20955701

Bone marrow stromal cells (MSCs) are being evaluated as a cellular therapeutic for immune-mediated diseases. We investigated the effects of MSCs in mice with chemically induced colitis and determined the effects of CD11b(+) cells based on the hypothesis that MSCs increase numbers of regulatory T cells.

Synthesis and in Vivo Imaging of a 18F-labeled PARP1 Inhibitor Using a Chemically Orthogonal Scavenger-assisted High-performance Method

Angewandte Chemie (International Ed. in English). Feb, 2011  |  Pubmed ID: 21328671

MRI with Magnetic Nanoparticles Monitors Downstream Anti-angiogenic Effects of MTOR Inhibition

Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. Apr, 2011  |  Pubmed ID: 20559742

To study the effect of mammalian target of rapamycin (mTOR) inhibition on angiogenesis with magnetic resonance imaging (MRI) using magnetic iron oxide nanoparticles (MNP).

Quantitating Antibody Uptake in Vivo: Conditional Dependence on Antigen Expression Levels

Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. Aug, 2011  |  Pubmed ID: 20809210

Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation.

Intraoperative Near-infrared Fluorescent Cholangiography (NIRFC) in Mouse Models of Bile Duct Injury: Reply

World Journal of Surgery. Mar, 2011  |  Pubmed ID: 20645091

Vasculitis: Molecular Imaging by Targeting the Inflammatory Enzyme Myeloperoxidase

Radiology. Jan, 2012  |  Pubmed ID: 22084204

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.

Bevacizumab with Angiostatin-armed OHSV Increases Antiangiogenesis and Decreases Bevacizumab-induced Invasion in U87 Glioma

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2012  |  Pubmed ID: 21915104

Bevacizumab (BEV) is an antiangiogenic drug approved for glioblastoma (GBM) treatment. However, it does not increase survival and is associated with glioma invasion. Angiostatin is an antiangiogenic polypeptide that also inhibits migration of cancer cells, but is difficult to deliver. Oncolytic viruses (OV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them and deliver anticancer agents to uninfected cells. We have tested a combination treatment of BEV plus an OV expressing angiostatin (G47Δ-mAngio) in mice-bearing human GBM. Using a vascular intracranial human glioma model (U87) in athymic mice, we performed histopathological analysis of tumors treated with G47Δ-mAngio or BEV alone or in combination, followed tumor response by magnetic resonance imaging (MRI), and assessed animal survival. Our results indicate that injection of G47Δ-mAngio during BEV treatment allows increased virus spread, tumor lysis, and angiostatin-mediated inhibition of vascular endothelial growth factor (VEGF) expression and of BEV-induced invasion markers (matrix metalloproteinases-2 (MMP2), MMP9, and collagen). This leads to increased survival and antiangiogenesis and decreased invasive phenotypes. We show for the first time the possibility of improving the antiangiogenic effect of BEV while decreasing the tumor invasive-like phenotype induced by this drug, and demonstrate the therapeutic advantage of combining systemic and local antiangiogenic treatments with viral oncolytic therapy.

PET/MRI of Inflammation in Myocardial Infarction

Journal of the American College of Cardiology. Jan, 2012  |  Pubmed ID: 22222080

The aim of this study was to explore post-myocardial infarction (MI) myocardial inflammation.

Extramedullary Hematopoiesis Generates Ly-6Chigh Monocytes That Infiltrate Atherosclerotic Lesions

Circulation. Jan, 2012  |  Pubmed ID: 22144566

Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6C(high) monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here, we hypothesized that the bone marrow outsources the production of Ly-6C(high) monocytes during atherosclerosis.

Bioorthogonal Reaction Pairs Enable Simultaneous, Selective, Multi-target Imaging

Angewandte Chemie (International Ed. in English). Jan, 2012  |  Pubmed ID: 22162316

Mutually orthogonal tetrazine-trans-cyclooctene and azide-cyclooctyne cycloaddition reactions were used simultaneously for the bioorthogonal labeling of two different live cell populations in the same culture. These small-molecule probes show good chemical reactivity and can be readily incorporated into biological systems.

Rapid Monocyte Kinetics in Acute Myocardial Infarction Are Sustained by Extramedullary Monocytopoiesis

The Journal of Experimental Medicine. Jan, 2012  |  Pubmed ID: 22213805

Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity's critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

Innate Response Activator B Cells Protect Against Microbial Sepsis

Science (New York, N.Y.). Feb, 2012  |  Pubmed ID: 22245738

Recognition and clearance of a bacterial infection are a fundamental properties of innate immunity. Here, we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA) B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern-recognition receptors, and produce granulocyte-macrophage colony-stimulating factor. Specific deletion of IRA B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.

Magnetic Nanoparticles and MicroNMR for Diagnostic Applications

Theranostics. 2012  |  Pubmed ID: 22272219

Sensitive and quantitative measurements of clinically relevant protein biomarkers, pathogens and cells in biological samples would be invaluable for disease diagnosis, monitoring of malignancy, and for evaluating therapy efficacy. Biosensing strategies using magnetic nanoparticles (MNPs) have recently received considerable attention, since they offer unique advantages over traditional detection methods. Specifically, because biological samples have negligible magnetic background, MNPs can be used to obtain highly sensitive measurements in minimally processed samples. This review focuses on the use of MNPs for in vitro detection of cellular biomarkers based on nuclear magnetic resonance (NMR) effects. This detection platform, termed diagnostic magnetic resonance (DMR), exploits MNPs as proximity sensors to modulate the spin-spin relaxation time of water molecules surrounding the molecularly-targeted nanoparticles. With new developments such as more effective MNP biosensors, advanced conjugational strategies, and highly sensitive miniaturized NMR systems, the DMR detection capabilities have been considerably improved. These developments have also enabled parallel and rapid measurements from small sample volumes and on a wide range of targets, including whole cells, proteins, DNA/mRNA, metabolites, drugs, viruses and bacteria. The DMR platform thus makes a robust and easy-to-use sensor system with broad applications in biomedicine, as well as clinical utility in point-of-care settings.

Origins of Tumor-associated Macrophages and Neutrophils

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22308361

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

The Neuropeptide Neuromedin U Promotes Autoantibody-mediated Arthritis

Arthritis Research & Therapy. Feb, 2012  |  Pubmed ID: 22314006

ABSTRACT: INTRODUCTION: Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary objective of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects. METHODS: Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU. RESULTS: NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux. CONCLUSIONS: NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.