Current Pharmaceutical Biotechnology - Volume 11, Issue 6, 2010

Over the last decade, the field of molecular imaging has witnessed tremendous expansion, partly owing to the rapid evolvement of imaging instruments dedicated to small animal studies which enabled non-invasive investigation of many biological events that could not be easily interrogated before these tools became available. This special issue of Current Pharmaceutical Biotechnology will cover a broad array of exciting topics in molecular imaging. Molecular imaging is a pivotal component of future “personalized medicine”. Positron emission tomography (PET) is the most sensitive imaging modality in the clinic. Several review articles in this special issue will focus on different aspects of PET imaging, including novel PET agents in clinical studies, recent development in PET instrumentation, and various methods to label proteins with 18F (the most-widely used PET isotope). A number of strategies have been employed to identify novel agents for molecular imaging applications. Among these, the use of Affibody molecules, development of new probes for melanoma imaging, identification of novel targeting ligands by phage display, and dual-targeted molecular probes for cancer imaging will be reviewed in this issue. Optical imaging is less expensive and more convenient than many other imaging modalities such as PET and magnetic resonance imaging (MRI). Therefore, it can serve as an attractive alternative in preclinical imaging studies since light penetration in small animals is less of a concern than in humans. This special issue will cover several topics in optical imaging such as new optical imaging systems, spectral fluorescence imaging, confocal fluctuation spectroscopy/imaging, imaging with Raman spectroscopy, as well as the recent development of quantum dot (i.e. fluorescent semiconductor nanoparticle) bioconjugates. Cell-based therapies hold enormous potential for treating many diseases and non-invasive imaging is an indispensable tool in monitoring the location and therapeutic efficacy of these cells. Two review articles in this special issue will be dedicated to two of the most intensively studied cell types, T-cells and human embryonic stem cells (hESCs), respectively. The future of “personalized medicine” would greatly benefit from the development of imaging biomarkers that can noninvasively monitor the therapeutic response. The development of agents that can image cell death (apoptosis and/or necrosis) in vivo, the current and future role of hyperpolarized 13CMRI, and the use of diffusion-weighted MRI for assessment of early cancer treatment response in many types of solid tumors, are also reviewed in this issue. Molecular imaging is an extremely dynamic field of research with far-reaching (potential) applications in clinical patient management. For example, 18F-FDG has been routinely used in cancer patient management and it is expected that many other new PET agents will be approved by the Food and Drug Administration (FDA) in the near future. Topics on molecular imaging can easily fill several text books and I sincerely hope that this special issue will provide a “sneak peak” of this highly interdisciplinary and fertile ground of research, which may lead to broad applications of these tools by scientists/clinicians from many different fields in answering their own biological questions of interest.

Molecular imaging represents an extremely important advancement in patient care and numerous agents have been developed and are being studied. Hundreds of molecular imaging agents currently exist for use in multiple modalities such as magnetic resonance imaging (MRI), computed tomography (CT), ultrasound, optical imaging, general nuclear medicine and positron emission tomography (PET). In PET, only a very small percentage of these agents are being used clinically in humans and more show promise for clinical use in the short-term future. The purpose of this paper is to present a brief overview of PET agents other than 18F-fluordeoxyglucose (FDG) that are either currently in clinical use in humans or in the authors' opinion are felt to show great promise for clinical use in the near future.

Positron emission tomography (PET) is used in the clinic and in vivo small animal research to study molecular processes associated with diseases such as cancer, heart disease, and neurological disorders, and to guide the discovery and development of new treatments. This paper reviews current challenges of advancing PET technology and some of newly developed PET detectors and systems. The paper focuses on four aspects of PET instrumentation: high photon detection sensitivity; improved spatial resolution; depth-of-interaction (DOI) resolution and time-of-flight (TOF). Improved system geometry, novel non-scintillator based detectors, and tapered scintillation crystal arrays are able to enhance the photon detection sensitivity of a PET system. Several challenges for achieving high resolution with standard scintillatorbased PET detectors are discussed. Novel detectors with 3-D positioning capability have great potential to be deployed in PET for achieving spatial resolution better than 1 mm, such as cadmium-zinc-telluride (CZT) and position-sensitive avalanche photodiodes (PSAPDs). DOI capability enables a PET system to mitigate parallax error and achieve uniform spatial resolution across the field-of-view (FOV). Six common DOI designs, as well as advantages and limitations of each design, are discussed. The availability of fast scintillation crystals such as LaBr3, and the silicon photomultiplier (SiPM) greatly advances TOF-PET development. Recent instrumentation and initial results of clinical trials are briefly presented. If successful, these technology advances, together with new probe molecules, will substantially enhance the molecular sensitivity of PET and thus increase its role in preclinical and clinical research as well as evaluating and managing disease in the clinic.

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. In particular, the interest in 18F-labeled proteins remains high for both diagnoses and therapy monitoring purposes. The development of labeling strategies for the synthesis of new 18F labeled protein is, however, not trivial. 18F-containing prosthetic groups are often required for protein labeling to obtain high yield under mild labeling conditions and keep the bioactive character of the proteins. This review highlights key aspects of protein 18F-labeling method and discussed representative examples including 18F-labeled human serum albumin, 18F-labeled Annexin V, 18F-labeled HER2 affibody, and 18F-labeled low density lipoprotein.

The current way to increase efficacy of cancer therapy is the use of molecular recognition of aberrantly expressed gene products for selective treatment. However, only a fraction of the patients have tumors with a particular molecular target. Radionuclide imaging of molecular targets might help to stratify patient for cancer treatment. Affibody molecules are scaffold proteins, which can be selected for high affinity recognition of proteinaceous molecular targets. The capacity to re-fold under physiological conditions allows labeling of Affibody molecules in a broad range of pH and temperatures with preserved binding properties. Peptide synthesis or introduction of a unique cysteine enables sitespecific labeling of Affibody molecules, resulting in uniform conjugates with well-defined pharmacological characteristics. The small size (7 kDa) of Affibody molecules provides rapid extravasation, rapid tumor penetration, and rapid clearance of unbound tracer from healthy organs and tissues. In combination with sub-nanomolar affinity, this results in high contrast in vivo imaging a few hours after injection. Excellent targeting has been demonstrated in pre-clinical studies with HER2-targeting Affibody molecules labeled with 99mTc and 111In for single photon computed tomography (SPECT), and 18F, 64Cu, 124I and 68Ga for positron emission tomography (PET). Pilot clinical data confirm the high potential of Affibody molecules.

Malignant melanoma represents a serious public health problem and is a deadly disease when it is diagnosed at late stage. Though 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) has been widely used clinically for melanoma imaging, other approaches to specifically identify, characterize, monitor and guide therapeutics for malignant melanoma are still needed. Consequently, many probes targeting general molecular events including metabolism, angiogenesis, hypoxia and apoptosis in melanoma have been successfully developed. Furthermore, probes targeting melanoma associated targets such as melanocortin receptor 1 (MC1R), melanin, etc. have undergone active investigation and have demonstrated high melanoma specificity. In this review, these molecular probes targeting diverse melanoma biomarkers have been summarized. Some of them may eventually contribute to the improvement of personalized management of malignant melanoma.

The advancement of molecular imaging hardware and disease targets has driven the demand of novel probes for diagnosis of human diseases. The high-throughput property of phage-display techniques makes the fast developments of these probes possible. The antibody and peptide phage display libraries have been widely used in ligand-selections for a variety of targets in tumors including osteosarcomas, pancreatic ductal adenocarcinomas, thyroid, head and neck squamous cell carcinomas, prostate, bladder, colon and gastric cancers. The phage display techniques also have been utilized in development of imaging agents for tumor therapy response, apoptosis, hypoxia, infection and inflammation, Alzheimer's diseases, diabetes and cardiovascular diseases.

Molecular imaging provides a powerful tool for early disease detection, characterization, and real-time monitoring of therapeutic responses. Central to molecular imaging is the development of imaging probes. Receptors that are overexpressed in tumors have been potential targets for cancer imaging using targeted molecular probes. Since many cancer types simultaneously overexpress several receptors, the design of dual receptor-targeted molecular imaging probes has recently been proposed. Peptide heterodimers, and microbubble-based dual-targeted probes have shown especially good performance for in vivo cancer imaging. The success of these molecular probes demonstrates that the dual-targeting strategy is generally a good approach for developing molecular probes. In this review, we will introduce the current state of development of dual-targeted molecular imaging probes and summarize the various dual-targeted molecular probes for in vivo cancer imaging.

Molecular imaging has become a research focus in recent years, which provides an effective information acquisition, analysis and processing methodology at cellular and molecular levels for biomedical study. As an important molecular imaging technique, optical molecular imaging, especially fluorescence and bioluminescence imaging, has attracted remarkable attention in tumor study and drug development for its excellent performance, non-radiativity and high costeffectiveness in comparison with conventional imaging modalities. Generally speaking, optical molecular imaging is regarded as the combination of traditional medical imaging technology and modern molecular biology, in which the advanced optics, biology, information, medicine, and other techniques are being married to non-invasively obtain in vivo physiological and pathological information sensitively, quantitatively, and specifically. Furhtermore, with the research of imaging theories, algorithms and molecular probes, optical imaging systems have been rapidly developed for biomedical study in molecular imaging discipline, including planar imaging systems, tomographic imaging systems, multimodality fusion systems and so on. This review focuses on some typical optical molecular imaging systems, especially for in vivo small animal use. It also provides a brief discussion on the future development and application of the optical molecular imaging systems.

Fluorescence-based molecular imaging in small animals is having a major impact on drug development and disease research and the ability to detect multiple molecular species at once is becoming increasingly important. Unlike bioluminescence, in fluorescence, ubiquitous autofluorescent signals from the skin need to be separated from that of labeled fluorophores to ensure proper quantitative data. Since its introduction in 2004, spectral imaging methods have become an important part of in vivo fluorescence imaging of small animals by enabling easy multiplexed imaging methods and through the quantitative removal of interfering skin autofluorescence signals. This article is a review of the literature on spectral imaging methods and applications in fluorescence imaging of small animals.

Currently, work with subnanomolar concentrations is routine while femtomolar and even single-molecule studies are possible with some efforts getting high on single-molecule biophysics and biochemistry. Methodological breakthroughs, such as reducing the background light contribution in single-molecule studies, which has plagued many studies of molecular fluorescence in dilute solution, and particularly in live cells, have recently described by us. We first demonstrated how optimized time-gating of the fluorescence signal, together with time-correlated single-photon counting, can be used to substantially boost the experimental signal-to-noise ratio about 140-fold, making it possible to measure analyte concentrations that are as low as 15 pM. By detection of femtomolar bulk concentrations, confocal microsopy has the potential to address the observation of one and the same molecule in dilute solution without immobilization or hydrodynamic/ electrokinetic focusing at longer observation times than currently available. We present relevant physics. The equations are derived using Einstein's approach showing how it fits with Fick's law and the autocorrelation function. An improved technology is being developed at ISS for femtomolar microscopy. The general concepts and provided experimental examples should help to compare our approach to those used in conventional confocal microscopy.

Raman spectroscopy, based on the inelastic scattering of a photon, has been widely used as an analytical tool in many research fields. Recently, Raman spectroscopy has also been explored for biomedical applications (e.g. cancer diagnosis) because it can provide detailed information on the chemical composition of cells and tissues. For imaging applications, several variations of Raman spectroscopy have been developed to enhance its sensitivity. This review article will provide a brief summary of Raman spectroscopy-based imaging, which includes the use of coherent anti-Stokes Raman spectroscopy (CARS, primarily used for imaging the C-H bond in lipids), surface-enhanced Raman spectroscopy (SERS, for which a variety of nanoparticles can be used as contrast agents), and single-walled carbon nanotubes (SWNTs, with its intrinsic Raman signal). The superb multiplexing capability of SERS-based Raman imaging can be extremely powerful in future research where different agents can be attached to different Raman tags to enable the interrogation of multiple biological events simultaneously in living subjects. The primary limitations of Raman imaging in humans are those also faced by other optical techniques, in particular limited tissue penetration. Over the last several years, Raman spectroscopy imaging has advanced significantly and many critical proof-of-principle experiments have been successfully carried out. It is expected that imaging with Raman Spectroscopy will continue to be a dynamic research field over the next decade.

Quantum dots (QDs) are new generation of fluorophores with superior optical properties. For biological applications of QDs, proper surface modification and further conjugation with biomolecules are necessary to make these nanocrystals biocompatible as well as target-recognizable. Preparation of QDs bioconjugates was reviewed in this paper to demonstrate general strategies in the bioconjugation of QDs and typical QDs bioconjugates including QDs conjugated with peptides, proteins or oligonucleotides were introduced. Recent examples on the applications of QDs bioconjugates for sensitive detection of biomolecules such as nucleic acids or proteins were reviewed. QDs bioconjugates used in cell labeling and trafficking, in detection of subcellular molecules and in imaging protein dynamics in live cells were also reviewed with an emphasis on current work reported in the past two years. Latest progress on the application of QDs bioconjugates for in vivo imaging was briefly covered and perspective on QDs bioconjugates and their applications in bioimaging was discussed with related to the issues to be addressed in future QDs applications.

The prospect of mobilizing the power of the immune system in the fight against cancer has fascinated generations of immunologists. After decades of sporadic successes scattered amongst frequent failures, the field of tumor immunology is now poised to take advantage of a greatly improved understanding of the molecular and cellular mechanisms of immune tolerance. Further enhancing the prospects of cancer immunotherapy are technological advantages that have resulted in sophisticated diagnostic and immune monitoring approaches. Amongst these approaches, molecular imaging has the potential to play a transformational role in the development of highly efficient immune therapies. This brief review outlines recent significant steps in this direction, and discusses strategies to incorporate molecular imaging in the design of the next generation of experimental and clinical cell-based immune interventions against cancer.

Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), and multimodality approaches. Although the focus of this review article is primarily on hESCs, the labeling/tracking strategies described here can be readily applied to other (stem) cell types as well. Non-invasive imaging can provide convenient means to monitor hESC survival, proliferation, function, as well as overgrowth (such as teratoma formation), which could not be readily investigated previously. The requirement for hESC tracking techniques depends on the clinical scenario and each imaging technique will have its own niche in preclinical/ clinical research. Continued evolvement of non-invasive imaging techniques will undoubtedly contribute to significant advances in understanding stem cell biology and mechanisms of action.

The noninvasive detection of cell death has significant diagnostic values. Molecular events in apoptosis and necrosis are a source of valuable surrogate markers for the detection of cell death. Two classes of imaging agents are being developed for imaging caspase activities and redistribution of membrane phospholipids, respectively. The current review looks at the molecular recognition mechanisms of existing and emerging agents in the physiological context of the surrogate markers. The imaging of caspase activities using substrate-derived agents has the advantage of high selectivity and can potentially allow the dissection of individual apoptotic pathways in vivo. The detection of membrane phospholipid redistribution using extracellular agents has the advantage of high target density and accessibility. The strength and limitations of each approach are discussed. Overall, in order to develop appropriate imaging techniques, it is important to understand the interactions between the agent and surogate markers on a molecular as well as physiological level. Such information is vital for fully appreciating the potential utilities of an imaging strategy.

Recent clinical practice for the management for cancer patients has begun to change from a statistical “one-size fits all” approach to medicine to more individualized care. Pre-treatment biomarkers (i.e. genetically and histologically based) have a growing role in providing guidance related to the appropriate therapy and likelihood of response; they do not take into account heterogeneity within the tumor mass. Thus, a biomarker which could be utilized to measure actual tumor response early following treatment initiation would provide an important opportunity to evaluate treatment effects on an individual patient basis. Diffusion weighted magnetic resonance imaging (DW-MRI) offers the opportunity to monitor treatment-associated alterations in tumor microenvironment using quantification of changes in tumor water diffusion values as a surrogate imaging biomarker. Results obtained thus far using DW-MRI have shown that changes in tumor diffusion values can be detected early following treatment initiation which correlate with traditional outcome measures. Sensitive imaging biomarkers are providing for the first time a means of assessing 3 dimensional tumor response early in the treatment cycle. This review highlights the development of DW-MRI and its proposed usefulness in the clinical management of cancer patients. The utility of DW-MRI for assessing therapeutic-induced response is further evaluated on tumors residing in the brain, head and neck and bone.

Hyperpolarized (HP) 13C labeled compounds can be used as MR contrast agents to investigate metabolic pathways in vivo in almost real time. To date, a high proportion of reported studies have utilized HP 1-13C pyruvate to investigate intracellular metabolism in tumors and other tissues. The long T1 relaxation time of the carboxylate carbon enables the 13C signal of the pyruvate to be followed for nearly 2 minutes following injection. During this time, pyruvate is rapidly metabolized to generate observable metabolites such as alanine and lactate. HP 13C labeled compounds have, for example, also been used to non-invasively probe physiological parameters such as pH, which emphasizes the expanding potential of the technique. The commercial availability of dynamic nuclear polarization (DNP) systems to generate hyperpolarized material for injection has made the technique available to researchers worldwide. As a consequence, DNP 13C MR has become a rapidly expanding area of research. The technique, with its specific strengths and weaknesses, has incredible potential coupled with inherent limitations, and this review aims to both present background to the technique and describe some of the necessary hardware and software essential to perform hyperpolarized 13C studies. An overview of the current and future role of HP 13C based molecular imaging is presented.