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Laboratory Automation In Vivo Molecular Imaging Multimodality Approaches Are Illuminating The goal of researchers using in vivo molecular imaging is to visualize biologic events in intact cells, organs, and whole organisms in their usual environment. Non-invasive techniques can provide the ability to follow biochemical processes, such as receptor-ligand and enzyme-substrate reactions, signal transduction, and developmental pathways as they occur in living cells. Using non-destructive techniques means that the processes can be followed over time in the same organism. Applications of molecular imaging technologies include furthering the study of normal development and pathogenesis, drug development, and testing therapeutic interventions.
Form and Function A significant portion of Thomas J. Meade’s research team at Northwestern University focuses on developing probes for magnetic resonance imaging (MRI) for in vivo molecular imaging. Meade, Professor, Chemistry Department, Northwestern University, Evanston, IL, observes that although “molecular imaging” may itself be a relatively new term, it derives from what used to be known as nuclear medicine, a concept that emerged about 40 years ago. Despite its age, however, says Meade, the field has taken its greatest strides in the last 10 years because of advances in the engineering aspect of MRI technology. Many laboratories choose to work within one imaging modality, though each has its limitations, says Meade. Positron emission tomography (PET), for example, has a sensitivity that “can’t be touched,” but its spatial and temporal resolutions are not as high as those seen in MRI. And, though MRI can yield the high levels of spatial and temporal resolutions necessary to see anatomic structures, Meade characterizes the current lack of probe sensitivity Vol. 45 ı No. 4 ı 2008
Detecting the activity of an enzyme by MR imaging. The complex is a caged probe that binds the lanthanide, Gd(III). The “roof” of the cage is an enzyme substrate for lacZ that, when present, removes the roof and turns the agent “on.” This allows for imaging gene expression in whole animals. Courtesy of Thomas J. Meade, Northwestern University, Evanston, IL.
as the “Achilles’ heel” of MRI. A goal of his research is to develop MRI probes that are more sensitive, and that can be activated by some biological process. Bioactivated MRI probes can potentially provide functional or physiological information along with structural information.
“Ultimately, basic science questions are driving the development of new probes that may have significant impact on translational clinical issues.” Bioactivated probes are activated by a physiologic event, for example, an enzyme reacting with its substrate. Because of their selectivity, these probes can be used to generate an image only when a particular event occurs. An example is the use of β-galactosidase to detect expression of the lacZ reporter sequence attached to a gene under study. The reaction is detected optically through the generation of a color reaction. In addition, Meade’s group has developed macrocyclic probes linking a sugar to magnetic resonance (MR) contrast agents. The agent is “silent” until β-galactosidase is activated by expression of the gene. Then, the location of the reaction can be pinpointed using MR.
Other contrast agents may be activated by a change in ion concentration, such as an influx of a cation due to a change in membrane potential in an active neuron. This could be exploited to improve on existing functional brain imaging technology, allowing for better imaging of brain function in real time. “We believe the future is dynamic, conditionally acting agents,” Meade says. Bioactivated probes could allow imaging of a living animal repeatedly over time, with specific probes designed to correlate gene expression with developmental events. Potential clinical applications include the ability to determine if therapeutic interventions end up in the right place. Probes could track pancreatic islet cells that are transplanted into the liver as a therapy for type 1 diabetes, or follow particles that are injected into blood vessels to cut off the blood supply to tumors. “Ultimately,” Meade observes, “basic science questions are driving the development of new probes that may have significant impact on translational clinical issues.”
Localization Alexei Bogdanov, Jr., Professor, Departments of Radiology and Cell Biology, University of Massachusetts, Worcester, MA, works in several areas of in vivo molecular imaging. One of www.biotechniques.com ı BioTechniques 375
Fluorescence resonance energy transfer (FRET) between near-infrared fluorochromes (NIRF) can be used to monitor experimental gene therapy to ensure that the appropriate gene is induced or silenced (A). Targeted imaging using a substrate designed for myeloperoxidase, an enzyme associated with inflammation and implicated in atherosclerosis due to the inflammatory component of vascular disease (B, C). Courtesy of Alexei Bogdanov, University of Massachusetts, Worcester, MA.
the challenges he sees in the field of molecular imaging—a challenge many other fields face as well—is how to deliver the imaging probes to the precise site of interest. “If there are any breakthroughs in [the] drug delivery field,” he says, “the field of molecular imaging will benefit from this immensely.” One area of interest Bogdanov describes as “futuristic” is looking at ways to image transcription factors in cells. An application in cancer is to elucidate abnormal signaling and gene expression using oligonucleotide duplex-linked probes that can bind to transcription factors to track cellular responses to protein binding. This technology, which is based on fluorescence resonance energy transfer (FRET) between near-infrared fluorochromes (NIRF) could ultimately be used to monitor experimental gene therapy to ensure that the appropriate gene is induced or silenced. Bogdanov, who primarily uses MR and optical imaging methods, says that his group is pursuing the use of small molecularmass paramagnetic and radioactive substrates, which they have developed in the recent past, as probes for enzyme activity in living animals. Bogdanov’s laboratory is aimed at imaging both enzymatic tags for targeted imaging, and endogenous enzymes. One of the developed substrates is designed for use with myeloperoxidase, an enzyme associated with inflammation and implicated in atherosclerosis due to the inflammatory component of vascular disease.
“If there are any breakthroughs in [the] drug delivery field, the field of molecular imaging will benefit from this immensely.”
Bogdanov observes that for clinical applications, imaging modalities that expose patients to little or no radiation are the most desirable. Because a patient can accumulate a combined high dose of X-ray radiation from computed tomography (CT) or high-energy γ radiation from PET, the number of repeat scans is limited for these technologies. Thus, there has been a recent push to replace CT with MR, where possible. Repeat scans over long periods of time are beneficial in that they could be used to monitor for disease and follow responses to treatment. Bogdanov’s group hopes to increase the sensitivity of MR to the level of other nuclear-medicine methods by using small molecular probes similar to those that have already been approved for human use, thus avoiding the use of radioisotopes.
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Green and Red Expression Jianghong Rao, a member of the Molecular Imaging Program, Stanford University, Stanford, CA, is developing molecular probes that can be used to monitor specific biologic processes under physiologic conditions. His lab is currently focusing on multimodality imaging of gene expression, RNA and RNA splicing, and protease function in vivo. Unlike Meade, who thinks of molecular imaging as a newer version of nuclear medicine, Rao sees the field as relatively new and not as well established as more traditional areas of research like biochemistry and biophysics. However, he acknowledges that molecular imaging has become more diverse during its development over the last 5 or 6 years due to the number of modalities that it encompasses, and has seen the increased use of non-invasive whole-body
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Concept of the ribozyme-based RNA imaging probe. Courtesy of Jianghong Rao, Stanford University, Palo Alto, CA.
optical imaging using fluorescence and bioluminescence. Rao notes that in addition to the original green fluorescent protein that has been used as probe, variants of many other colors are available, including red. He also notes that near-infrared (NIR) fluorescent proteins are in development for optical imaging. This will be an important tool, he observes, because longer-wavelength probes are preferred for whole animal imaging. Rao thinks bioluminescence offers significant advantages over fluorescence for whole-animal imaging. It has low background and high signal-to-noise ratios, and most organisms do not normally have many bioluminescent proteins of their own (some exceptions include fireflies and some marine animals). Currently, bioluminescent probes emit in the blue to yellow range, and Rao notes that it’s a challenge to move the emission into the red and near-infrared region. His group is engineering bioluminescence into the red range, taking advantage of incorporating quantum dots (fluorescent semiconductor nanoparticles) to serve as acceptors in bioluminescence energy resonance transfer (BRET). The BRET donor is a mutant Renilla luciferase (Luc8), and the resulting quantum dot conjugate is “self-illuminating:” that is, it can emit light without external photon excitation. Rao’s group has achieved emission in the 800 nm range, which overcomes the short wavelength problem. The probe also overcomes the problem of background fluorescence seen with unmodified quantum dots. With further conjugation to a tumor-targeting ligand,
this agent may be used to detect tumor cells. Other applications of such modified quantum dots include sensing and imaging of protease and kinase functions. It is also possible to visualize RNA and RNA splicing activity in vivo using ribozyme splicing activity linked to a reporter. Rao’s group is developing strategies to image low copy number mRNA transcripts in cells by amplifying the signal, but it is also interested in direct detection methods so that mRNA can be imaged in real time in whole animals. By attaching an antisense sequence of the mRNA of interest to the 5′′ end of a trans-splicing ribozyme and a bioluminescent reporter (for example, firefly luciferase) to the 3′′ end, they designed a ribozyme-based RNA reporter. The reporter recognizes the mRNA of interest and generates a fusion mRNA reporter where the activity level is directly tied to the level of mRNA of interest. This method also allows the imaging of siRNA inhibition of target gene expression in vivo. “Ideally, we want to improve the system to look at low level expression and look at several mRNAs at time,” says Rao. “The next step is a challenge.” Two different color reporters could be used to detect two mRNAs in the same organism or cell, he says.
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- Lynne Lederman is a freelance medical writer in Mamaroneck, NY.
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