Emerging concepts in molecular MRI David E Sosnovik and Ralph Weissleder

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1 Emerging concepts in molecular MRI David E Sosnovik and Ralph Weissleder Molecular magnetic resonance imaging (MRI) offers the potential to image some events at the cellular and subcellular level and many significant advances have recently been witnessed in this field. The introduction of targeted MR contrast agents has enabled the imaging of sparsely expressed biological targets in vivo. Furthermore, high-throughput screens of nanoparticle libraries have identified nanoparticles that act as novel contrast agents and which can be targeted with enhanced diagnostic specificity and range. Another class of magnetic nanoparticles have also been designed to image dynamic events; these act as switches and could be used in vitro, and potentially in vivo, as biosensors. Other specialized MR probes have been developed to image enzyme activity in vivo. Lastly, the use of chemical exchange and off-resonance techniques have been developed, adding another dimension to the broad capabilities of molecular MRI and offering the potential of multispectral imaging. These and other advances in molecular MRI offer great promise for the future and have significant potential for clinical translation. Addresses Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston MA, USA Corresponding author: Sosnovik, David E (sosnovik@nmr.mgh.harvard.edu) This review comes from a themed issue on Analytical biotechnology Edited by Joseph Wu and Jagat Narula Available online 28th November /$ see front matter # 2006 Elsevier Ltd. All rights reserved. DOI /j.copbio Introduction The attributes of magnetic resonance imaging (MRI; see Box 1) allow high-resolution anatomical and functional images of numerous organ systems to be obtained and also make it highly suited to the molecular imaging mission, namely the imaging of events at the cellular and subcellular level [1,2,3]. The detection of events at this level, however, often requires nanomolar sensitivity, thus precluding the use of conventional gadolinium chelates as molecular MR imaging agents, as they display only micromolar sensitivity. The principal response to the inadequate sensitivity of conventional gadolinium chelates has been to synthesize novel MR contrast agents with significantly higher relaxivities [3]. These include paramagnetic gadolinium-containing liposomes [4,5] or micelles [6,7], and superparamagnetic iron oxide nanoparticles [8,9]. The use of iron oxide nanoparticles for liver imaging [10], lymph node characterization [11], diagnosis of tissue inflammation [12,13,14], and stemcell labeling [15,16] is becoming fairly well established, and will thus not be further discussed in this review. Several novel techniques and applications in molecular MRI have emerged in the past few years and will be highlighted here. These include the in vivo imaging of sparsely expressed biological targets using targeted MR contrast agents, the surface modification of nanoparticles to yield nanoparticle libraries with enhanced target specificity, the use of iron oxide containing magnetic relaxation switches as in vitro and in vivo biosensors, in vivo imaging of enzyme activity with activatable MR contrast agents, and the development of CEST and PARACEST techniques that allow multispectral proton MRI to be performed. Cardiovascular disease remains the leading cause of mortality in industrialized societies and applications in this area will thus be highlighted, although the techniques listed above are applicable to a broad range of disease states. Particular, but not exclusive, attention will also be paid to applications involving iron oxide nanoparticles. The interested reader is referred to other articles in this volume for a more detailed discussion on other nanoparticles and nanostructures. Finally, given the scope of this review, the discussion will be limited to proton MRI. Targeted MR contrast agents Fibrin-targeted MR contrast agents were amongst the first molecular MR probes to be developed, and exploited the high levels of fibrin expressed within thrombi. These agents were successfully used to detect both arterial and venous thrombi in a variety of models [5,17,18]. Recently, however, more sparsely expressed targets such as surface integrins [19], surface phospholipids on apoptotic cells [20 ], vascular adhesion molecules [21 ], and macrophage receptors [7], amongst others, have been imaged non-invasively in vivo. The detection of a sparsely expressed surface target, particularly one within an organ parenchyma or an atherosclerotic plaque, ideally requires the MR contrast agent to have a long half-life in the blood, the ability to penetrate the tissue of interest, and the ability to detect and amplify the biological signal of interest. The superparamagnetic iron oxides, in particular, fulfill many of these criteria [22]. Cross-linked iron oxide (CLIO) is a highly stabilized form of monodisperse iron oxide or MION [9,22]. The

2 Molecular MRI Sosnovik and Weissleder 5 Box 1 MRI technology and its attributes. Magnetic resonance imaging (MRI) is a non-invasive diagnostic technique that has the potential to image some events at the cellular or subcellular level. This imaging technology is based on the interaction of protons with each other and with surrounding molecules in a tissue of interest. When placed in a strong magnetic field protons precess or rotate at a given frequency and are able to accept energy from a radiofrequency wave applied at this rotational or resonance frequency. The behavior of the energy inserted in to the system is described by two relaxation constants: the T2 or transverse relaxation time and the T1 or longitudinal relaxation time. Different tissues have different relaxation times, and this can be used to produce endogenous contrast between different tissues. Exogenous contrast agents can further enhance tissue contrast by selectively shortening either the T1 or T2 in a tissue of interest. The MR image can be weighted to detect differences in either T1 or T2 by adjusting parameters in the acquisition. MRI offers several advantages over other imaging modalities. Firstly, it is non-ionizing as it detects the magnetic signals generated by protons and other molecules. The technique is also tomographic, enabling any tomographic plane through a three-dimensional volume to be imaged. High-resolution images with excellent soft tissue contrast between different tissues can be obtained. Lastly, multiple contrast mechanisms are possible using MRI and the technique can be used to provide anatomical as well as physiological readouts. cross-links on CLIO are aminated, allowing a large variety of ligands to be conjugated to the nanoparticle with a high degree of stability and relative ease. Near-infrared fluorochromes can be attached to the amine groups to form a dual modality magnetofluorescent nanoparticle [23]. Thereafter, many copies of the targeting ligand can be attached to the nanoparticle to form a multivalent (i.e. more than one copy of the targeting ligand) magnetofluorescent nanoparticle. Recently reported examples of two such ligands include annexin for apoptosis imaging [24] and a phage-derived peptide specific for the adhesion molecule VCAM-1 [21,25 ]. The properties of the magnetofluorescent annexin probe, AnxCLIO-Cy5.5, have been previously described [24]. Cardiomyocyte apoptosis in a mouse model of ischemia-reperfusion was successfully imaged with this probebymriin vivo [20 ]. Preliminary results with the probe also suggest that it can successfully detect even the low levels of cardiomyocyte apoptosis seen in heart failure. The adhesion molecule VCAM-1 is expressed by damaged endothelium early in the atherosclerotic process and was successfully imaged in the aortic roots of apoe / mice in vivo (Figure 1) [21 ]. In addition, the beneficial effect of statin therapy on VCAM-1 expression could be documented with the probe in vivo by demonstrating decreased probe accumulation in the statin-treated mice [21 ]. Thus, this VCAM-1-sensing iron oxide probe not only demonstrated adequate sensitivity to detect a sparsely expressed molecular marker, but also exhibited an adequate dynamic range to detect a treatment effect. The therapeutic effect of the anti-angiogenic agent fumagillin has also been demonstrated in a rabbit model of vascular injury with a gadolinium-containing liposome targeted to the a V B 3 integrin. This integrin is a marker of angiogenesis, inflammation and instability within an atherosclerotic plaque [26 ]. Although, larger than iron oxide probes, the hyperpermeability of vessels involved in plaque angiogenesis allows the gadolinium-containing liposome to penetrate the plaque to enable imaging of new vessel formation. Gadolinium-containing immunomicelles have also recently been developed and, when decorated with an antibody to the macrophage scavenger receptor, have been shown by ex vivo MRI to detect plaque macrophages [7]. In the near future it is likely that superparamagnetic iron oxides with even higher relaxivities will be generated [27]. Further advances are also likely to be seen in the synthesis of novel gadolinium chelates and constructs. The imaging of a wide variety of sparsely expressed molecular targets by MRI in small animal models will thus become an established technique in molecular biology and pharmaceutical development. Targeted molecular MRI in large animal models will require ligand synthesis to be scaled up to support this, and will thus require careful selection. Further translation into the clinical realm will require even more careful consideration, as each new construct will require a completely separate profile of pharmacokinetic and toxicity screening. Nevertheless, the translation of a few well-selected targeted iron oxide probes into the clinical realm remains highly likely. Iron oxide nanoparticle libraries Although the clinical translation of targeted MR contrast agents will require careful selection of the most appropriate agents, this restriction will not apply in the preclinical arena. In this realm, high-throughput screening and synthesis techniques are likely to be used to generate a diverse array of nanoparticles with small surface modifications. Minor modification of the surface of the iron oxide nanoparticle CLIO-Cy5.5 has already been shown to drastically alter its cellular uptake [28 ]. In addition, these modifications can now be performed quickly and at room temperature using convenient click chemistry techniques [29]. The generation of a library of nanoparticles for macrophage imaging has recently been described [28]. In its baseline state, CLIO-Cy5.5 has a high affinity for both resting and activated macrophages. Conjugation of the chemical moiety succinimidyl iodo acetate (SIA) to the aminated sidechains on CLIO-Cy5.5, however, almost completely abolished its uptake by any macrophages whatsoever. High-throughput screening also revealed CLIO conjugates with specific affinity for either resting or activated macrophages: the agent CLIO-Gly, for

3 6 Analytical biotechnology Figure 1 In vivo MRI of VCAM-1 expression in apoe / mice. The dotted white line marks the short axis plane of the aortic root, used in the remainder of the images. MR imaging at 9.4 Tesla (a) before and (b) after the injection of a VCAM-1-targeted magnetofluorescent probe reveals a significant change in signal intensity in the aortic root plaque. The accumulation of the probe in the endothelium of the aortic root and its specificity for VCAM-1 are confirmed by (c) fluorescence microscopy and (d) immunohistochemistry. (Figure adapted from [21 ] with permission.) instance, was found to be taken up only by activated macrophages (Figure 2), whereas the agent CLIO-bentri was found to be avid for only resting macrophages [28 ]. Given the potential of stem-cell therapy in a wide variety of diseases, it is likely that large nanoparticle libraries will be developed to aid the preclinical understanding of these cells. Probes specific for cell type, activation and differentiation will be developed. The use of such nanoparticle libraries could facilitate the development of effective stem-cell therapies and a better understanding of their regenerative potential. MR-detectable nanoswitches and biosensors The targeted molecular MR contrast agents, described above, are generally designed to image targets with a fairly constant level of expression during the image acquisition. However, another class of magnetic nanoparticles, designed to image dynamic events, has recently been described [30]. These magnetic relaxation switches consist of iron oxide nanoparticles that undergo reversible assembly and disassembly in the presence of a specific enzyme or chemical compound, producing a change in transverse magnetic relaxivity. Magnetic relaxation switches have previously been shown to be capable of detecting proteases [30], oligonucleotides [31], viral particles [32], and entantiomeric impurities [33] in solution. The synthesis of magnetic relaxation switches specific for a wide variety of diagnostic markers used in clinical medicine is certainly feasible, and could prove a useful addition to currently used in vitro diagnostic techniques. Magnetic relaxation switches have also recently been used to determine the concentration of analytes, such as glucose [34 ] and calcium [35], in solution. A construct consisting of CLIO-glucose and concavalin-a was able to accurately determine glucose concentration over the clinically meaningful range [34 ]. In the presence of lower glucose concentrations, the CLIO-glucose conjugates bind to concavalin-a and form aggregates with higher relaxivities. However, as the glucose concentration increases, the CLIO-glucose conjugates are displaced from concavalin-a and the degree of nanoparticle aggregation decreases [34 ]. This concept is demonstrated in

4 Molecular MRI Sosnovik and Weissleder 7 Figure 2 Figure 3 Generation of an iron oxide nanoparticle library for macrophage sensing. The parent compound CLIO-NH 2 is taken up by both resting and activated macrophages. Activation of the macrophages by granulocyte macrophage colony stimulating factor (GMCSF), oxidized LDL (OxLDL) or lipopolysaccharide (LPS) produces a significant increase in avidity for the compound CLIO-Gly, which is otherwise minimally taken up by resting macrophages. Conversely, the agent CLIO-bentri is taken up by resting macrophages, but not by activated macrophages. (a) Bar chart showing the degree of uptake of the different iron oxide nanoparticles. The y axis shows the fluorescence on uptake in arbitrary units (au). (b) Near-infrared fluorescence microscopy of probe uptake (color scale) fused with phase contrast images of the macrophage populations. (Figure adapted from [28 ] with permission.) Figure 3. The ability of this construct to accurately measure glucose concentration across a semipermeable membrane suggests it could find use as an implantable biosensor in the future [34 ]. It should also be possible for a library of biosensors to be included within the semipermeable membrane, allowing a panel of metabolites to be sensed continuously, simultaneously and noninvasively by MRI in vivo [36 ]. In vivo MRI of enzyme activity Numerous situations exist where an enzyme of interest is expressed only locally or within a solid tissue and, in such cases, systemic detection within a semipermeable Magnetic nanosensing of glucose concentrations. (a) In the presence of low glucose concentrations the CLIO-glucose nanoparticles (red) bind to concavalin-a (green) and form clusters. (b) In the presence of high glucose (brown) concentrations, CLIO-glucose is displaced from concavalin-a causing the disassembly of the nanoswitch. (c,d) Size distribution of nanoparticles by light scattering. At baseline (red) the majority of nanoparticles are between nm in size, increase to nm after the addition of concavalin-a (brown) and then return to their baseline size distribution (yellow) after the addition of glucose to the solution. (e,f) Changes in glucose concentration can be sensed by detecting changes in the transverse relaxation time (T2), induced by assembly or disassembly of the nanoswitch. (Figure adapted from [34 ] with permission.) membrane is inadequate. The use of magnetic relaxation switches to image local enzyme activity, however, is complicated by aggregation of the nanoparticles into lysosomes when they are not protected by a semipermeable membrane [37]. The concept of enzyme-mediated aggregation, however, has recently been extended to novel gadolinium conjugates, which are significantly less sensitive to nonspecific local aggregation [38,39 ]. Myeloperoxidase is an enzyme involved in free radical generation and is thought to have a central role in atherosclerotic plaque instability. The peptide serotonin, when oxidized by this enzyme, tends to aggregate into dimers and oligomers. A gadolinium-serotonin chelate, capable of sensing myeloperoxidase activity in vivo, has thus been developed [38]. Once the serotonin moiety in the chelate is activated by myeloperoxidase, the probe

5 8 Analytical biotechnology Figure 4 with an offset in their Larmor frequencies, a concept which forms the basis of CEST, PARACEST and LIPO- CEST imaging [41,42,43 ]. The protons in a targeted liposome, for instance, containing a particular paramagnetic lanthanide will have a resonance frequency that is offset from the surrounding bulk protons [42 ]. In the presence of magnetization transfer across the liposome, selective excitation at this offset frequency results in a detectable change in the bulk water signal. If two or more probes, directed against distinct molecular targets, have sufficiently different offset frequencies this can allow multispectral proton MRI of more than one target to be performed by sequential selective excitation [44 ]. In vivo MRI of myeloperoxidase activity in mice. (a) Only a transient increase in signal intensity is seen when the conventional gadolinium chelate, gadolinium-diethylenetriaminepentaacetic acid (Gd-DPTA), is given to mice with myositis. (b) A sustained increase in signal intensity is seen when the gadolinium-serotonin chelate is used. (c) Human myeloperoxidase embedded into a matrix gel and implanted into the right flank of a mouse was also able to activate the gadolinium-serotonin probe. Implanted matrix gel without myeloperoxidase (left flank) did not activate the probe. (Figure adapted from [39 ] with permission.) assembles into dimers and quatromers that tend to be retained locally and which have a significantly higher longitudinal relaxivity than the parent compound [38,39 ]. The presence of myeloperoxidase in a mouse model of myositis has been successfully imaged in vivo with this probe [39 ], as shown in Figure 4. Multispectral and off-resonance MRI One of the traditional limitations of molecular imaging by MRI has been its ability to provide only a single composite readout of proton behavior. This is in contrast to single-photon emission computed tomography (SPECT) and fluorescence techniques, which in addition to being highly sensitive are also multispectral and thus capable of providing several simultaneous readouts of individual probes. One potential solution to this limitation is to combine proton MRI with, for instance, fluorine MRI [40 ]. Because the Larmor (or precession) frequencies of protons and fluorine are reasonably close, the imaging of both signatures can usually be performed on the same scanner. An alternative approach, however, is to exploit the magnetization transfer between two pools of protons The concept of selectively imaging protons shifted off the central resonance frequency has now also been extended to the imaging of iron oxide probes. A variety of offresonance techniques have been developed, all of which result in positive contrast in the vicinity of the iron oxide [45,46 ]. In vivo experience with these off-resonance and CEST-based techniques, however, is quite limited, although an initial CEST study in humans with brain tumors has recently been reported [47]. While highly promising, further experience will be needed with these techniques to fully judge their true potential. Conclusions Conventional MR contrast agents currently rely on changes in probe biodistribution or pharmacokinetics in disease states. Future MR contrast agents, however, will probe anatomy at the cellular and subcellular level and will either be directed against specific targets or activated by specific enzymes. In addition, the same probes could form the basis of MR-detectable biosensors. Thus, molecular MRI is likely to continue to grow in importance in the future and will strongly complement the central role of MRI in conventional anatomical imaging. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Weissleder R: Molecular imaging in cancer. Science 2006, 312: This is an excellent review of molecular imaging in cancer but the issues discussed (early diagnosis of disease, drug development, personalized management of disease and the challenges involved in obtaining clinical approval for a novel agent) apply broadly. 2. Weissleder R: Molecular imaging: exploring the next frontier. Radiology 1999, 212: Sosnovik D, Weissleder R: Magnetic resonance and fluorescence based molecular imaging technologies. Prog Drug Res 2005, 62: Lanza GM, Abendschein DR, Yu X, Winter PM, Karukstis KK, Scott MJ, Fuhrhop RW, Scherrer DE, Wickline SA: Molecular imaging and targeted drug delivery with a novel,

6 Molecular MRI Sosnovik and Weissleder 9 ligand-directed paramagnetic nanoparticle technology. Acad Radiol 2002, 9(Suppl 2):S330-S Winter PM, Caruthers SD, Yu X, Song SK, Chen J, Miller B, Bulte JW, Robertson JD, Gaffney PJ, Wickline SA et al.: Improved molecular imaging contrast agent for detection of human thrombus. Magn Reson Med 2003, 50: Frias JC, Williams KJ, Fisher EA, Fayad ZA: Recombinant HDL-like nanoparticles: a specific contrast agent for MRI of atherosclerotic plaques. J Am Chem Soc 2004, 126: Lipinski MJ, Amirbekian V, Frias JC, Aguinaldo JG, Mani V, Briley-Saebo KC, Fuster V, Fallon JT, Fisher EA, Fayad ZA: MRI to detect atherosclerosis with gadolinium-containing immunomicelles targeting the macrophage scavenger receptor. Magn Reson Med 2006, 56: Shen T, Weissleder R, Papisov M, Bogdanov A Jr, Brady TJ: Monocrystalline iron oxide nanocompounds (MION): physicochemical properties. Magn Reson Med 1993, 29: Wunderbaldinger P, Josephson L, Weissleder R: Crosslinked iron oxides (CLIO): a new platform for the development of targeted MR contrast agents. Acad Radiol 2002, 9(Suppl 2):S304-S Harisinghani MG, Jhaveri KS, Weissleder R, Schima W, Saini S, Hahn PF, Mueller PR: MRI contrast agents for evaluating focal hepatic lesions. Clin Radiol 2001, 56: Harisinghani MG, Barentsz J, Hahn PF, Deserno WM, Tabatabaei S, van de Kaa CH, de la Rosette J, Weissleder R: Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N Engl J Med 2003, 348: Dunn EA, Weaver LC, Dekaban GA, Foster PJ: Cellular imaging of inflammation after experimental spinal cord injury. Mol Imaging 2005, 4: Turvey SE, Swart E, Denis MC, Mahmood U, Benoist C, Weissleder R, Mathis D: Noninvasive imaging of pancreatic inflammation and its reversal in type 1 diabetes. J Clin Invest 2005, 115: Very nice demonstration of the use of a long circulating magnetofluorescent iron oxide probe to diagnose and monitor tissue inflammation. 14. Kraitchman DL, Heldman AW, Atalar E, Amado LC, Martin BJ, Pittenger MF, Hare JM, Bulte JW: In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003, 107: Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R: Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000, 18: Hill JM, Dick AJ, Raman VK, Thompson RB, Yu ZX, Hinds KA, Pessanha BS, Guttman MA, Varney TR, Martin BJ et al.: Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003, 108: Botnar RM, Perez AS, Witte S, Wiethoff AJ, Laredo J, Hamilton J, Quist W, Parsons EC Jr, Vaidya A, Kolodziej A et al.: In vivo molecular imaging of acute and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent. Circulation 2004, 109: Botnar RM, Buecker A, Wiethoff AJ, Parsons EC Jr, Katoh M, Katsimaglis G, Weisskoff RM, Lauffer RB, Graham PB, Gunther RW et al.: In vivo magnetic resonance imaging of coronary thrombosis using a fibrin-binding molecular magnetic resonance contrast agent. Circulation 2004, 110: Winter PM, Morawski AM, Caruthers SD, Fuhrhop RW, Zhang H, Williams TA, Allen JS, Lacy EK, Robertson JD, Lanza GM et al.: Molecular imaging of angiogenesis in early-stage atherosclerosis with alpha(v)beta3-integrin-targeted nanoparticles. Circulation 2003, 108: Sosnovik DE, Schellenberger EA, Nahrendorf M, Novikov MS, Matsui T, Dai G, Reynolds F, Grazette L, Rosenzweig A, Weissleder R et al.: Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle. Magn Reson Med 2005, 54: The testing and application of a MR-detectable apoptosis-sensing probe is described. Probe validation is performed in cell culture, in tissue phantoms and in a mouse model in vivo. The authors demonstrate that targeted iron-oxide probes are able to penetrate the capillary endothelium and bind to a specific target within a tissue of interest. The ability to image a targeted MR contrast agent in the myocardium in vivo with MRI is shown, and the value of a dual modality (MRI and fluorescence) imaging approach is demonstrated. 21. Nahrendorf M, Jaffer FA, Kelly KA, Sosnovik DE, Aikawa E, Libby P, Weissleder R: Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis. Circulation 2006, 114: Comprehensive molecular imaging paper ranging from the use of phage display to identify a targeting ligand to the demonstration of a treatment effect by in vivo molecular imaging. The authors describe the synthesis and testing of a VCAM-1 sensing probe and its ability to image atherosclerotic plaques in apoe knockout mice in vivo. The ability of the probe to detect early atheroma in the mouse in vivo, detect human plaques ex vivo and monitor a treatment effect in the mouse in vivo is shown. 22. Wunderbaldinger P, Josephson L, Weissleder R: Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjug Chem 2002, 13: Kircher MF, Mahmood U, King RS, Weissleder R, Josephson L: A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res 2003, 63: Schellenberger EA, Sosnovik D, Weissleder R, Josephson L: Magneto/optical annexin V, a multimodal protein. Bioconjug Chem 2004, 15: Kelly KA, Nahrendorf M, Yu AM, Reynolds F, Weissleder R: In vivo phage display selection yields atherosclerotic plaque targeted peptides for imaging. Mol Imaging Biol 2006, 8: Useful article on the use of phage display to detect ligands specific to a target or cell of interest. 26. Winter PM, Neubauer AM, Caruthers SD, Harris TD, Robertson JD, Williams TA, Schmieder AH, Hu G, Allen JS, Lacy EK et al.: Endothelial alpha(v)beta3 integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler Thromb Vasc Biol 2006, 26: Important article demonstrating the principle of a dual diagnostic and therapeutic agent. A liposome targeted to the alphav/beta 3 integrin is used to image and treat pathological angiogenesis in a rabbit model of aortic atheroma. The liposome contains both gadolinium for MR imaging and the anti-angiogenic agent fumagillin, which is released locally after ligand binding. 27. Moffat BA, Reddy GR, McConville P, Hall DE, Chenevert TL, Kopelman RR, Philbert M, Weissleder R, Rehemtulla A, Ross BD: A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI. Mol Imaging 2003, 2: Weissleder R, Kelly K, Sun EY, Shtatland T, Josephson L: Cellspecific targeting of nanoparticles by multivalent attachment of small molecules. Nat Biotechnol 2005, 23: A library of nanoparticles is generated in this study by modifying the surface of a nanoparticle with small molecules. The different compounds in the library show markedly different affinities for resting and activated macrophages. The generation of these sorts of libraries is likely to become both more important and more common in the preclinical setting. 29. Sun EY, Josephson L, Weissleder R: Clickable nanoparticles for targeted imaging. Mol Imaging 2006, 5: Perez JM, Josephson L, O Loughlin T, Hogemann D, Weissleder R: Magnetic relaxation switches capable of sensing molecular interactions. Nat Biotechnol 2002, 20: Perez JM, O Loughin T, Simeone FJ, Weissleder R, Josephson L: DNA-based magnetic nanoparticle assembly acts as a magnetic relaxation nanoswitch allowing screening of DNA-cleaving agents. J Am Chem Soc 2002, 124: Perez JM, Simeone FJ, Saeki Y, Josephson L, Weissleder R: Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media. J Am Chem Soc 2003, 125:

7 10 Analytical biotechnology 33. Tsourkas A, Hofstetter O, Hofstetter H, Weissleder R, Josephson L: Magnetic relaxation switch immunosensors detect enantiomeric impurities. Angew Chem Int Ed Engl 2004, 43: Sun EY, Weissleder R, Josephson L: Continuous analyte sensing with magnetic nanoswitches. Small 2006, 2: A highly visionary paper describing the use of magnetic nanoswitches to sense analyte concentrations across a semi-permeable membrane. In particular, the ability of this technology to accurately measure glucose concentrations is studied. 35. Atanasijevic T, Shusteff M, Fam P, Jasanoff A: Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin. Proc Natl Acad Sci USA 2006, 103: Sun EY, Josephson L, Kelly KA, Weissleder R: Development of nanoparticle libraries for biosensing. Bioconjug Chem 2006, 17: An extension of the prior work from this group. Surface modification of iron oxide nanoparticles is used to generate a library of nanoparticles for analyte sensing. 37. Hogemann-Savellano D, Bos E, Blondet C, Sato F, Abe T, Josephson L, Weissleder R, Gaudet J, Sgroi D, Peters PJ et al.: The transferrin receptor: a potential molecular imaging marker for human cancer. Neoplasia 2003, 5: Chen JW, Pham W, Weissleder R, Bogdanov A Jr: Human myeloperoxidase: a potential target for molecular MR imaging in atherosclerosis. Magn Reson Med 2004, 52: Chen JW, Querol Sans M, Bogdanov A Jr, Weissleder R: Imaging of myeloperoxidase in mice by using novel amplifiable paramagnetic substrates. Radiology 2006, 240: Excellent paper showing the ability to image enzyme activity in vivo with an activatable MRI probe. The activity of the enzyme myeloperoxidase is successfully imaged with a gadolinium-serotonin chelate. Exposure of the chelate to the enzyme causes it to undergo polymerization and increase its longitudinal relaxivity. 40. Caruthers SD, Neubauer AM, Hockett FD, Lamerichs R, Winter PM, Scott MJ, Gaffney PJ, Wickline SA, Lanza GM: In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 Tesla. Invest Radiol 2006, 41: Paper dealing with the performance of both proton and fluorine MRI. Liposomes containing gadolinium and a perfluorocarbon are shown to be visible at either frequency. 41. Zhou J, Payen JF, Wilson DA, Traystman RJ, van Zijl PC: Using the amide proton signals of intracellular proteins and peptides to detect ph effects in MRI. Nat Med 2003, 9: Aime S, Delli Castelli D, Terreno E: Highly sensitive MRI chemical exchange saturation transfer agents using liposomes. Angew Chem Int Ed Engl 2005, 44: The concept of LIPOCEST is discussed in this paper. 43. Woods M, Woessner DE, Sherry AD: Paramagnetic lanthanide complexes as PARACEST agents for medical imaging. Chem Soc Rev 2006, 35: Very good review article on PARACEST imaging. 44. Aime S, Carrera C, Delli Castelli D, Geninatti Crich S, Terreno E: Tunable imaging of cells labeled with MRI-PARACEST agents. Angew Chem Int Ed Engl 2005, 44: The concept of multispectral or tunable proton MRI is demonstrated in this paper. Rat hepatoma cells loaded with either Europium or Terbium can be separately and selectively visualized. 45. Cunningham CH, Arai T, Yang PC, McConnell MV, Pauly JM, Conolly SM: Positive contrast magnetic resonance imaging of cells labeled with magnetic nanoparticles. Magn Reson Med 2005, 53: Off resonance imaging of superparamagnetic iron oxides is described in this paper. 46. Mani V, Briley-Saebo KC, Itskovich VV, Samber DD, Fayad ZA: Gradient echo acquisition for superparamagnetic particles with positive contrast (GRASP): sequence characterization in membrane and glass superparamagnetic iron oxide phantoms at 1.5T and 3T. Magn Reson Med 2006, 55: An alternative technique for off resonance imaging is described. 47. Jones CK, Schlosser MJ, van Zijl PC, Pomper MG, Golay X, Zhou J: Amide proton transfer imaging of human brain tumors at 3T. Magn Reson Med 2006, 56: