Angelique Y. Louie

In vivo visualization of gene expression using magnetic resonance imaging

High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, β-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.

Multimodality Imaging Probes: Design and Challenges

The conundrum of modality selection in clinical diagnostic imaging is that modalities with the highest sensitivity have relatively poor resolution, while those with high resolution have relatively poor sensitivity. In recent years, the idea of using multiple modalities in conjunction has gained in popularity and researchers have come to realize that the complementary abilities of different imaging modalities could be harnessed to great effect by using them in tandem. The idea of combining imaging technologies moved to the mainstream with the advent of the first successful commercial fused instruments. The first fused PET/CT instrument, developed in 1998 by Townsend and colleagues in collaboration with Siemens Medical, was available commercially in 2001. The “Biograph” was named as one of the “Inventions of the Year” in 2000 by Time magazine, and its success was such that by 2003 fused PET/CT instruments were available from all of the major clinical instrument manufacturers, GE, Philips, CTI, and Siemens.1 Over the ensuing years, PET/CT sales increased with such vigor that by the year 2006 there were virtually no sales of standalone PET instruments; all PET sales were as part of multimodality systems.2–4 The next wave of innovation has been in PET/MRI-fused instruments, which have generated much hope for improved patient safety and imaging capability over PET/CT. Although research on PET/MRI instruments was initiated around the same time as PET/CT, the economic and engineering challenges of merging the two modalities slowed development, and the first commercial PET/MRI prototype for a human scale hybrid scanner was not unveiled until 2007.5,6 With hybrid technology clearly on the rise, the excitement over these new instruments has triggered a tumult of activity in probe design and development as investigators seek to boost the clinical benefits of hybrid instrument technology. n nAs the preponderance of recent reviews and increase in attention at scientific meetings will attest, there has been a surge in research on multimodal contrast agent development over the past few years.5,7–17 For molecular imaging, particularly, the rise in multimodal instrumentation has sparked hopes for new ways to track multiple molecular targets simultaneously, or to use different imaging methods in combination to more clearly delineate localization and expression of biochemical markers. In the best of situations, the combined imaging methods and probes work synergistically to allow high-resolution, high-sensitivity investigation of biological activity. For example, with dual function probes for PET/MRI, the high sensitivity of PET can be used as a whole body screen to identify regions of interest, thereby reducing the volume of tissue that needs to be scanned; this reduces scan time required for high-resolution imaging by MRI.5,18 However, probe design and development has sometimes preceded the identification of clear applications that merit use of the multimodal principle. There are many literature examples of probes that are “all dressed up with nowhere to go”; they possess unique physical properties that have yet to find a clear province in medicine or biology. Nonetheless, it is not unusual for technology to sometimes presage the need, and it is these advances that can spur imaginative solutions to problems that had been intractable with the previously existing technology. The goal of multimodal functionality has already reaped benefits by driving innovation in many areas of chemical synthesis, most notably in nanotechnology. n nWhile combining multimodal detectability in the same probe is not necessitated by all applications, there can be advantages to this arrangement. A single probe helps to ensure the same pharmacokinetics and colocalization of signal for each modality if that is a concern. It also can avoid putting the additional stress on the body’s blood clearance mechanisms that can accompany administration of multiple doses of agents. The caveat is that because the sensitivities of different imaging modalities can vary by 3 orders of magnitude, it may not be practical to simply add all functionalities to one molecule, although we will see that is a common design, because the requirements for contrast agent concentrations can be vastly different between modalities. In this review, we provide an overview of the many strategies that have been applied to achieve multimodal functionality in a single probe unit. These span the range from small molecule to nanoparticulate systems and vary in complexity from facile encapsulation or conjugation of commercially available probes to de novo synthesis. This review is limited to reports from the last approximately 5 years that deal with agents that carry two or more species of contrast enhancers. Tables summarizing the physical properties from cited articles are included with each major category of probe to facilitate “browsing” by the reader.

Receptor mediated uptake of a radiolabeled contrast agent sensitive to β-galactosidase activity

Radiolabeling of the MRI contrast agent 1-[2-(beta-galactopyranosyloxy)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane with (111)In, and its evaluation is reported. Radiolabeling was performed in acetate buffer with 50-78% radiochemical yield. In vitro studies revealed that the asialoglycoprotein receptor-poor cell line MH1C1 has low uptake, while the receptor-rich cell lines BNL-CL2 and Hep G2 have higher uptake. In vivo, the uptake of the compound in receptor-rich organ liver was very high. Blocking the receptor in vivo, reduced liver uptake by 90% suggesting that the compound localizes in receptor-enriched tissues by binding to the asialoglycoprotein receptor.

Design and Characterization of Magnetic Resonance Imaging Gene Reporters

We review the current status for magnetic resonance contrast agents that are designed to act as gene reporters. The basic concepts behind magnetic resonance and contrast enhancement are discussed, and factors that influence design of activatable contrast agents are presented. Several designs for magnetic resonance imaging (MRI) gene reporters are described, including contrast agents that are activated by beta-galactosidase, and marker genes that code for proteins that sequester iron. Methods to characterize the uptake and delivery of contrast agents are outlined.

30 – Mapping Gene Expression by MRI

This chapter describes the use of magnetic resonance (MR) techniques for imaging gene expression. Magnetic resonance contrast agents are typically composed of chelated paramagnetic ions. A paramagnetic ion in solution can act as a transceiver of radiation. The unpaired electrons residing on the ion form a permanent magnetic dipole with a magnitude dependent on the number of unpaired spins. Several contrast agents are developed that are sensitive to a number of biologically relevant targets such as pH, Ca 2+ concentration, and enzyme activity. Unlike existing contrast agents that constantly enhance signal, these agents behave in a conditional fashion and do not act until cleaved by the product of the lacZ gene, the enzyme β-galactosidase. These MR contrast agents are based on the framework of a clinical contrast agent, Gd(HP-DO3A), that has been modified with a carbohydrate “cap” that blocks access of water to the gadolinium. The introduction of exogenous genes (transgenes) for gene therapy or for generating transgenic animals requires expression at high levels and is a better candidate for detection by magnetic resonance imaging.

Fluorescence resonance energy transfer: FRET studies of ligand binding to cell surface receptors

We describe a simple optical system employing fluorescence resonance energy transfer (FRET) to identify potential binding domains on the macrophage scavenger receptor for the ligand maleylated bovine serum albumin (mal-BSA). Using a plasma membrane vesicle system, we placed donor probes on the ligand and acceptor probes in the membrane to determine the distance of bound ligand from the cell surface. Two donors and three acceptors were employed. Transfer between ligand covalently modified with multiple dansyl molecules and hexadecanoylaminoeosin in the membrane yielded a distance of 46.5 ± 7.5 å; transfer from the same type of donors to octadecylrhodamine B in the membrane gave a distance of 58.5 ± 3.0 å. No transfer was observed between ligand mono-labeled with fluorescein and l,l′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate in the membrane. This suggests that the orientation of mal-BSA bound to the receptor places the fluorescein probe too far from the lipid surface to experience energy transfer. The distance information identifies a potential location for the binding site, which can be compared to structural information about the receptor and used to extract a binding sequence.

Fluorescence energy transfer studies on the macrophage scavenger receptor

The macrophage scavenger receptor is a transmembrane, trimeric glycoprotein which recognizes a number of negatively charged ligands. Cross competition studies of various ligands indicate that the scavenger receptor may bear more than one type of binding site or that there may be more than one type of receptor. In this study we employed resonance energy transfer techniques to identify the location of the binding site for maleylated bovine serum albumin. Using vesicles derived from plasma membrane, we labeled the ligand with a donor probe and labeled the membrane surface with acceptor probes to determine the distance of bound ligand from the membrane surface. Measurements were taken with three different donor-acceptor pairs. Transfer measurements for ligand labeled with dansyl and HAE (hexadecanoylaminoeosin) as the acceptor yielded a distance of 47 angstrom from the surface of the plasma membrane. Similar measurements employing the same donors but using ORB (octadecylrhodamine B) as the acceptor produced a distance of 58 angstrom. Assuming that the receptor extends perpendicularly from the cell surface this distance lies within the two receptor `domains closest to the cell surface. These domains include the spacer region, with no distinct proposed structure and a region which has sequence similarity to an alpha helical coiled coil. No transfer was observed between ligand monolabeled with fluorescein and DiI in the membrane. This suggests that the orientation of mal-BSA bound to receptor places the fluorescein probe too far from acceptor on the membrane surface to experience energy transfer.