A Terbium-Based PARACEST MR Contrast Agent for in Vivo Imaging Beyond the MT Effect
Todd C. Soesbe¹, Federico A. Rojas-Quijano¹, A. Dean Sherry^1,2
1Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, TX, United States; ²Department of Chemistry, The University of Texas at Dallas, Dallas, TX, United States

Chemical exchange saturation transfer (CEST) agents create contrast in MR images by exchanging their saturated lanthanide bound protons with unsaturated bulk water protons. CEST agents can be selectively activated by applying a 2 to 10 second long frequency-specific saturation pulse, tuned to the bound proton frequency, just before imaging. Chemical exchange of the saturated bound protons with bulk water leads to a reduced water signal and darkening in the MR image. These agents hold great potential to further extend the functional and molecular imaging capabilities of MR. Some published applications include measuring tumor pH, angiogenesis, and the tissue distribution of glucose and other metabolites. CEST agent bound proton frequencies are typically shifted 5 to 50 ppm from bulk water (0 ppm). Unfortunately, this is the same range of the in vivo Magnetization Transfer (MT) effect. The MT effect arises from dipolar exchange of protons with endogenous tissue materials such as macromolecules and cell membranes. The MT effect typically spans from ±100 ppm (relative to bulk water) and is proportional to saturation pulse power. As a consequence, the contrast produced by the CEST agent can be totally masked by the tissue MT effects, which greatly complicates in vivo imaging. In an effort to avoid the MT effect and enhance in vivo CEST imaging, our group has recently developed a Tb3+-based paramagnetic CEST (PARACEST) agent with an unusually long bound water exchange lifetime. The bound proton frequency for this agent is at -600 ppm, which is far outside the normal tissue MT window. Although other Tb3+-based PARACEST agents have been reported, this agent’s slower water exchange rate allows for an order of magnitude reduction in saturation pulse power, making it more suitable for in vivo studies. We present in vitro images of our Tb3+-based PARACEST agent to demonstrate its potential for in vivo imaging without the requirement of subtracting out tissue MT contributions.

Molecular imaging aims to detect the presence and spatial distribution of specific biomarkers in tissue. However, for many diseases the detection of these biomarkers must be done at very low concentrations to maximize diagnostic and prognostic value. Due to lack of sensitivity in conventional MRI techniques, exogenous contrast agents (e.g. SPIO, PARACEST) are being widely studied to lower concentration detection thresholds. Recently, targeted hyperpolarized xenon-based biosensors that exploit the exchange of solvated 129Xe between bulk solution (XeW) and cryptophane-A (CryA) molecular cages (XeC) have demonstrated high sensitivity (1). To build upon this work, a filamentous bacteriophage M13 was chosen as a scaffold upon which a large number of CryA copies could be assembled. M13 bacteriophage are routinely employed in phage display techniques used in panning for targeting moieties such as single chain fragment antibodies (scFv) (2), and thus can be straightforwardly targeted to biomarkers allowing for drastically increased CryA payloads per bound target. The purpose of this study was to investigate the feasibility of using an M13 bacteriophage modified with cryptophane-A molecular cages as a sensitive xenon-based MR contrast agent and to determine the detection thresholds of CryA-modified phage.

Multi-Color in Vivo MR Imaging of Lymph Nodes Using DIACEST Liposomes
Guanshu Liu^1,2, Matthew M. Moake³, Assaf A. Gilad^2,4, Muksit Jamil², Yah-el Har-el², Chris Long³, Piotr Walczak^2,4, Jiangyang Zhang², Amanda Cardona², Marco A. DeLiso², George Sgouros², Jeff W.M. Bulte^2,4, Peter C.M. van Zijl^1,2, Michael T. McMahon^1,2
1F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, MD, United States; ²Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, United States; ³Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States; ⁴Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States

We developed a new MR-visible liposome system based on labeling with three distinct diamagnetic Chemical Exchange Saturation Transfer (DIACEST) agents, L-arginine, poly-L-lysine and glycogen. Using saturation frequency swept MRI with B0-correction, the accumulation of all three types of DIACEST liposomes in mouse popliteal lymph nodes could be visualized. As a proof of concept, we demonstrate the first in-vivo multi-contrast (multi-color) MRI using two DIACEST agents, L-arginine liposomes and poly-L-lysine liposomes, that were simultaneously injected to two footpads of the same mouse. This new system allows direct monitoring of liposomal uptake in lymph nodes without any paramagnetic or super-paramagnetic contrast material.

Targeting the Endothelial Cell Surface: Novel Transgenic Mice for Molecular Imaging of Vascular Development
Cesar Augusto Berrios-Otero¹, Benjamin B. Bartelle¹, Anne E. Friedland¹, Daniel H. Turnbull^1,2
1Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States; ²Department of Radiology, New York University School of Medicine, New York, United States

Paramagnetic contrast agents targeted to cell membrane receptors or other surface proteins are currently of great interest for molecular imaging with MRI. A potential problem with current targeting methods is the limited targeting efficiency, which combined with the low sensitivity of many paramagnetic agents can severely compromise the application of these approaches for in vivo imaging.  One way to circumvent problems in targeting contrast agents to surface receptors is to increase the binding affinity of the ligand to its target. An intriguing possibility is to take advantage of the high binding affinity of avidin and biotin. In the current study, transgenic mice expressing an engineered biotin ligase (BirA) and a cluster of biotinylation substrate sequences (Biotags) fused to a transmembrane protein domain were generated. Expression was driven by a minimal Tie2 promoter-enhancer, providing high transgene levels during angiogenesis in developing mouse embryos. Targeting was tested in embryos by means of intracardiac injections of an Avidin-Gd based T1-agent and high resolution 3D T1-weighted imaging.

Targeted Imaging of EGF Receptor Expression in Gli36 Tumor Xenografts Using Monoclonal Antibody Conjugates
Mohammed Salman Shazeeb^1,2, Christopher Howard Sotak^1,3, Alexei Bogdanov^3
1Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, United States; ²Graduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester, MA, United States; ³Department of Radiology, University of Massachusetts Medical School, Worcester, MA, United States

Targeted EGF receptor imaging in Gli36 tumor xenografts implanted in the rat brain was achieved using monoclonal antibody (mAb) conjugates that facilitate local binding of a paramagnetic molecular substrate diTyr-DTPA(Gd) at the EGFR expression sites.  Following mAb conjugate administration, diTyr-DTPA(Gd) was retained for a significantly longer period of time as compared to the administration of the contrast agent without mAb conjugate pre-treatment.  The increased retention of diTyr-DTPA(Gd) following mAb conjugate administration is consistent with enzyme-mediated coupling of the paramagnetic agent to EGFR-overexpressing cells in the tumor; allowing effective MRI visualization of conjugate co-localization at the targeted site.

In Vivo 3D ¹⁹F Fast Spectroscopic Imaging (F-UTSI) of Angiogenesis on Vx-2 Tumors in Rabbits Using Targeted Perfluorocarbon Emulsions
Rolf Lamerichs¹, Muhammed Yildirim^1,2, Aart J. Nederveen³, Jaap Stoker³, Gregory M. Lanza⁴, Samuel A. Wickline⁴, Shelton D. Caruthers^4
1Philips Research, Eindhoven, Netherlands; ²Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands; ³Department of Radiology, Academic Medical Center, Amsterdam, Netherlands; ⁴Washington University, St Louis, MO, United States

Quantitative molecular MRI of angiogenesis using site-targeted ¹⁹F agents has great potential.  Many ¹⁹F agents, however, possess complex spectra with many resonances over a wide ppm range.  Fluorine ultra-fast Turbo Spectroscopic Imaging (F-uTSI) has been developed to overcome these drawbacks while offering the advantage of distinguishing various ¹⁹F compounds based on chemical shift differences thereby allowing ‘multi-color’ imaging.  Herein, F-uTSI is shown to be an efficient, sensitive technique for quantitatively detecting minute amounts of ¹⁹F contrast agents in vivo while overcoming the confounding problems associated with chemical shift.  Employing functionalized perfluorocarbon nanoparticles in Vx2 tumor-bearing rabbits, angiogenic maps were created.

Uptake of a Fibrin-Targeted Contrast Agent Could Direct Therapy Following Deep Vein Thrombosis
Marcelo E. Andia¹, Prakash Saha², Andrea J. Wiethoff¹, Ulrike Blume¹, Tobias Schaeffter¹, Alberto Smith², Rene M. Botnar^1
1Division of Imaging Sciences, Kings College London, London, United Kingdom; ²Academic Department of Surgery, Cardiovascular Division, Kings College London, London, United Kingdom

The in vivo evaluation of the stage of organization or resolution of venous thrombosis could lead the medical treatment decision in venous thrombosis diseases. In this work we show that the use of EP-2104R, a fibrin specific contrast agent could give valuable information of the stage of thrombus resolution in an in vivo animal model.

Targeted Iron Oxide Particles for in Vivo MR Detection of Atherosclerotic Lesions Using Antibodies Against Oxidized Low Density Lipoprotein: Effect of Particle Size
Karen Catherin Briley-Saebo¹, Sung Kee Ryu², Simone Green², Venkatesh Mani³, Stephen Dickson³, Sotirios Tsimikas², Zahi A. Fayad^3
1Radiology and Gene and Cell Medicine, Mount Sinai School of Medicine, NY, United States; ²Vascular Medicine, University of California San Diego, La Jolla, CA, United States; ³Radiology, Mount Sinai School of Medicine, NY, United States

Oxidized low-density lipoproteins (OxLDL) play a major role in plaque progression. Although OxLDL-targeted gadolinium micelles have been used for in-vivo detection of intraplaque macrophages, safety issues may limit clinical utility. The aim of the current study was to evaluate the in-vivo efficacy of oxLDL-targeted iron oxides. Small (<25nm) and large (>50nm) oxLDL-targeted particles were administered (4-mgFe/kg) to ApoE-/- mice. Imaging was performed 24 hours p.i. at 9.4T. Significant enhancement (ƒ´R2*>50%) was observed for the small oxLDL-targeted particles. Untargeted and large formulations exhibited limited enhancement. This study suggests that small OxLDL-targeted particles may allow for safe detection of foam cells.

Molecular MRI of Myocardial Angiogenesis After Acute Myocardial Infarction
Marlies Oostendorp¹, Kim Douma¹, Allard Wagenaar¹, Jos MGM Slenter¹, Tilman M. Hackeng¹, Marc AMJ van Zandvoort¹, Mark J. Post¹, Walter H. Backes^1
1Maastricht University Medical Centre (MUMC+), Maastricht, Netherlands

Here, a molecular MRI method is presented to non-invasively image angiogenic activity in vivo in a murine model of myocardial infarction using cyclic cNGR-labeled paramagnetic quantum dots (pQDs). The tripeptide cNGR homes specifically to CD13, an aminopeptidase that is strongly upregulated during myocardial angiogenesis. cNGR-QDs allowed specific detection of post-infarction myocardial angiogenesis, as shown by the strong contrast observed in the infarcted mouse heart on molecular MRI, and by the colocalization of cNGR-pQDs with vascular endothelial cells as detected by fluorescence microscopy.

Optimization of Liposomal Theragnosis: Quantitative T₁ Measurement of Drug Distribution and Release in Deep-Seated Tumor Using Multimodal Thermo-Sensitive Polymer-Modified Liposome
Daisuke Kokuryo¹, Seiji Nakashima², Kai-Hsiang Chuang³, Iwao Kanno¹, Kenji Kono², Ichio Aoki^1
1Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan; ²Graduate School of Engineering, Osaka Prefecture University, Sakai, Osaka, Japan; ³Singapore Bioimaging Consortium, Singapore, Singapore

A multimodal thermo-sensitive polymer-modified liposome (MTPL) loaded with anticancer drugs and contrast agents would be a powerful 'Theragnostic (therapy + diagnosis)' tool. In this paper, drug concentration in deep-seated tumor was evaluated using MTPL and a rapid quantitative imaging technique. Heat-triggered drug-release from MTPL was visualized in combination with the temperature distribution. MTPL concentration in the tumor area was maintained for between 4 and 12 hours after administration. We concluded that to minimize side-effects the optimum time to apply a heat-trigger is 12 - 24 hours after MTPL administration.