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Magnetic and fluorescent nanoparticles for multimodality imaging Willem JM Mulder1†, Arjan W Griffioen2, Gustav J Strijkers3, David P Cormode1,4, Klaas Nicolay3,5 & Zahi A Fayad1,6 †Author

for correspondence Sinai School of Medicine, Imaging Science Laboratories, Department of Radiology, One Gustave L Levy Place, New York, NY 10029, USA Tel.: +1 212 241 7717; E-mail: willem.mulder@ mountsinai.org 2Maastricht University, Angiogenesis Laboratory, Research Institute for Growth and Development, Department of Pathology/Internal Medicine, University Hospital, PO Box 5800, 6202 AZ Maastricht, The Netherlands Tel.: +31 433 874 630; E-mail: aw.griffioen@ path.unimaas.nl 3Eindhoven University of Technology, Biomedical NMR, Department of Biomedical Engineering, PO Box 513, 5600 MB Eindhoven, The Netherlands Tel.: +31 402 473 727; E-mail: [email protected] 4Tel.: +1 212 241 6858; Email: david.cormode@ mountsinai.org 5Tel.: +31 402 475 789; E-mail: [email protected] 6Tel.: +1 212 241 6858; E-mail: zahi.fayad@ mssm.edu 1Mount

Keywords: fluorescence imaging, fluorescence microscopy, intravital microscopy, iron oxide nanoparticles, liposomes, magnetic resonance imaging, micelles, molecular imaging, multimodality imaging, nanoparticles, optical imaging, quantum dots, whole-body photonic imaging part of

The development of nanoparticulate contrast agents is providing an increasing contribution to the field of diagnostic and molecular imaging. Such agents provide several advantages over traditional compounds. First, they may contain a high payload of the contrast-generating material, which greatly improves their detectability. Second, multiple properties may be easily integrated within one nanoparticle to allow its detection with several imaging techniques or to include therapeutic qualities. Finally, the surface of such nanoparticles may be modified to improve circulation half-lives or to attach targeting groups. Magnetic resonance imaging and optical techniques are highly complementary imaging methods. Combining these techniques would therefore have significant advantages and may be realized through the use of nanoparticulate contrast agents. This review gives a survey of the different types of fluorescent and magnetic nanoparticles that have been employed for both magnetic resonance and optical imaging studies.

Imaging technologies, such as computed tomography (CT), ultrasound and magnetic resonance imaging (MRI), have traditionally been employed to visualize anatomical structures and to assess the physiological function of tissue and organs [1]. In the last decade, the focus of research in imaging techniques has shifted towards the visualization of (patho)physiological processes at the cellular and molecular level, primarily in the research setting. This is the emerging domain of molecular imaging, which can be defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level through the use of imaging devices [1,2]. Sensitive imaging techniques, including nuclear methods such as positron-emission tomography (PET) and single photon-emission computed tomography (SPECT), are the main imaging modalities that have been reported for use in molecular imaging; however, these techniques have a relatively low spatial resolution and lack anatomical definition. MRI is a diagnostic tool that is characterized by its ability to generate 3D images of opaque and soft tissue with relatively high spatial resolution [3,4]. Apart from anatomical information, metabolic and functional parameters can also be obtained with MRI. Contrast in anatomical MR images is mainly due to differences in proton density and inherent differences in the relaxation times of tissue water. Importantly, relaxation can be manipulated with the use of exogenous contrast agents and, in the clinical setting, contrast agents are currently applied in

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30–40% of clinical MRI scans [5]. Due to the attractive properties of the MRI modality, molecular imaging contrast agents that are MRI active have been developed by a number of research groups since the mid-1990s. Nevertheless, in order for MRI to become a competitive molecular imaging modality, the problem of the inherent low sensitivity associated with this technique must be addressed, which may be realized by developing contrast agents with a very high relaxivity [3,6–8]. Progress in generating suitably high relaxivities has been achieved primarily through the formulation of nanoparticulate agents containing high payloads of gadolinium [9–12] or iron oxide [12]. Due to recent developments in chemistry and nanotechnology, the successful synthesis of more effective versions of these agents has been achieved, and so MRI is becoming an increasingly important molecular imaging technique. Significantly, multimodal MRI contrast agents have been developed that are equipped with labels for complementary imaging modalities, such as fluorescence [13–16]. Imaging tools highly complementary to MRI are optical techniques [17,18], such as confocal microscopy, intravital microscopy [19] and fluorescence imaging [18,20,21]. These techniques allow the detection of multiple fluorescent species with high speed and sensitivity, ranging from cellular resolution at small scanning windows to whole-body imaging. The major limitation of optical methods, however, is related to the low tissue penetration depth of light and, Nanomedicine (2007) 2(3), 307–324

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therefore, these techniques are mostly employed ex vivo on excised tissues. Nevertheless, in vivo fluorescence techniques, such as intravital microscopy and fluorescence imaging, are becoming increasingly important and with the development of new techniques, such as nearinfrared (NIR) optical imaging, the penetration has improved from 1–2 mm to several centimeters [22]. In addition to the aforementioned limitations, a drawback of these optical techniques is that little anatomical information can be obtained. Therefore, the combination of MRI and optical methods would provide significant advantages [13] and can be accomplished by the design of probes that exhibit both magnetic and fluorescent properties. This can be realized with nanotechnology, which offers the exciting possibility of creating sensitive, targeted and macromolecular contrast agents that have multiple properties integrated [23]. The first part of this review briefly introduces the MR and optical imaging technologies and will discuss their capabilities and limitations. This is followed by a section on the pathophysiological parameters that can be used for imaging. Subsequently, the main focus of this review will be on the reported developments and applications of a wide range of multimodal nanoparticulate agents that are employed for parallel MR and optical imaging. Examples include dextran-coated iron oxide particles conjugated with fluorescent molecules [24] and lipidic nanoparticles, such as liposomes [11], microemulsions and micelles, that are equipped with both magnetic and fluorescent amphiphiles [25,26]. Furthermore, the latest developments of endogenous virus [27] and lipoprotein [28] agents that have been modified for multimodality imaging and nanoparticles based on fluorescent quantum dots (QDs) with integrated magnetic properties [29] will be discussed. Although the main focus will be on the aforementioned fluorescent and magnetic probes, combinatory agents for other imaging modalities and therapy will also be highlighted.

3D images of opaque tissue with high spatial resolution and is diagnostically used in neurology, but also in oncology and cardiology. Using clinical scanners, voxel resolutions in the submillimeter range can be achieved while, experimentally, at high magnetic field strengths, isotropic voxel resolutions down to 25 µm have been accomplished in vivo. The MRI signal arises from the water protons of tissues that are placed in the magnetic field of the MR scanner. This magnetic field causes a net nuclear magnetization that can be disturbed by a radio frequency pulse. Intrinsic regional differences in the rate of return to the original thermal equilibrium, a process known as relaxation, can be exploited to generate the contrast in MR images. There are two principal relaxation processes: spin–lattice or longitudinal relaxation, characterized by a so-called T1 relaxation time; and spin–spin or transverse relaxation defined by the T2 parameter. The transverse relaxation process may be accelerated by macroscopic and microscopic magnetic field inhomogeneities, in which case the latter relaxation process is referred to as T2* relaxation. The intrinsic relaxation times of tissue water are dependent on the physiological environment and may be altered in pathological tissue. Importantly, exogenous paramagnetic and superparamagnetic materials shorten the relaxation times of water and can be used, in combination with the appropriate imaging parameters, to generate contrast [3]. For magnetic resonance molecular imaging purposes, this is the main strategy that has been employed, although other principles, such as chemical exchange saturation transfer agents that can be activated to generate contrast, have also been used [32]. Also, instead of MRI based on the proton signal and contrast enhancement with materials that increase proton relaxation rates, the direct MR visualization of fluorine-containing probes is currently also under investigation [33,34]. This allows probe detection without background signal with the use of 19F MRI.

Magnetic resonance & optical imaging techniques Magnetic resonance imaging MRI is among the most versatile imaging methods available in both clinical and research settings, since it excels in the visualization of soft tissues and because extensive methods to generate contrast for tissue differentiation have been developed [30,31]. MRI is capable of generating

Limitations of magnetic resonance imaging

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Although MRI excels in anatomical definition, resolution and possibilities to generate contrast compared with other clinically available imaging methods, molecular imaging with this technique needs thorough validation, since the signal observed might originate from nonspecific accumulation of the applied probe. In future science group

Magnetic and fluorescent nanoparticles for multimodality imaging – REVIEW

addition, issues such as nonspecific interaction, the fate of the agent once it has interacted with its target, its biodistribution and the uptake by organs such as the liver, spleen, or kidneys need to be investigated in detail in order to truly understand the interactions of the applied agent in the body. Another important feature that has to be considered is the fact that a signal increase or decrease within heterogeneous tissue has to be detected. This requires an MRI scan before the application of the agent, which must be compared with the image after application of the agent. A complementary technique that enables the validation of the MRI findings has to meet the following criteria: the technique should be able to detect the probe with high sensitivity, without significant background signal, but with the possibility of simultaneously marking other important features, such as cell type, the expression of genes, or the upregulation of (cell surface) proteins. Optical fluorescence techniques meet these requirements and can be employed ex vivo on excised tissue but also in vivo [35] with techniques such as intravital microscopy [19] and fluorescence imaging [17,18,20]. We will only briefly discuss these techniques, as well as their possibilities, as the topic has been recently discussed in depth elsewhere [13,18,20,36]. Immunofluorescence techniques

One of the primary utilities of optical techniques in combination with magnetic MRI is the validation and interpretation of the MRI findings as well as probe localization and its cellular fate [37]. Immunofluorescence staining on excised tissue can be performed, for example, which allows the simultaneous visualization of different markers. While cell nuclei are usually stained with 4´,6-diamidino-2-phenylindole (DAPI), which binds to DNA, specific cell types, such as endothelial cells, macrophages or tumor cells, and/or cellular targets can be visualized using fluorescently labeled antibodies. As well as the investigation of target tissue, other tissues and organs can be investigated for probe localization and probe fate with fluorescence microscopy. This is especially important when a probe’s clearance by the reticuloendothelial system or the kidneys must be investigated. When conducting targeted imaging studies, one must keep in mind that the percentage of the intravenously injected dose that reaches the selected target usually never exceeds 10–15% [38]. future science group

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Intravital microscopy

In addition to fluorescence microscopy performed on ex vivo specimens, in vivo microscopy studies, usually referred to as intravital microscopy studies [19], have also been performed. In principle, there is little difference between the optical configuration of conventional microscopy methods and intravital microscopy. Nevertheless, the technique investigates a living animal instead of a tissue slice, which evidently requires very different properties of the microscopic setup. Multiphoton microscopy techniques, such as two-photon, have particularly high potential for in vivo imaging. Two-photon microscopy is especially suited for the in vivo imaging of thick specimens, since the NIR radiation used in twophoton excitation has several orders of magnitude less absorption in biological specimens than ultraviolet (UV) or blue-green light, enhancing the penetration depth [39]. Although most studies have employed intravital microscopy techniques on tumors, neurological [39] and also cardiovascular [40] applications are under investigation. Whole-body photonic imaging

A third optical technique that has received a lot of attention and has undergone great progress in the last few years is whole-body photonic imaging [36]. This technique allows bioluminescence and fluorescence whole-body imaging of small animals. It is both sensitive and fast and recent developments in NIR probes and tomographic methods allow the reconstruction of 3D images of optical contrast with penetration depths in the centimeter to decimeter range [20]. The resolution of the technique is in the millimeter to submillimeter range and, using spectral differentiation, it is possible to simultaneously visualize multiple species. Multimodality imaging approaches

The combination of the different imaging modalities discussed above, in particular due to their complementary features, offers an attractive approach to investigate molecular and cellular processes, tissue characteristics, such as vessel density and permeability, and morphological changes of pathological tissues. This multimodal screening of pathology has been established and rapidly advanced primarily in animal models, but future technological advances will generate possibilities for clinical use. The success of this combinatory approach and the applicability of 309


the different techniques greatly depend on the availability of probes that have optimized properties for their detection with the different imaging modalities. After a concise section that deals with pathophysiology, an overview of probes for dual-modality imaging and their applications will be provided. Utility of pathophysiological parameters for imaging There are several markers that are associated with pathological processes and can be exploited for MRI and optical-enhanced imaging in combination with suitable probes. For example, many diseased sites are characterized by enhanced permeability of blood vessels [41], as highlighted in the following examples. During tumor development, new blood vessels are formed, a process that is referred to as angiogenesis [42]. These newly formed vessels are immature and leaky and therefore facilitate the nonspecific leakage and accumulation of macromolecular materials at such sites [43]. Many other pathologies, such as atherosclerosis, rheumatoid arthritis, endometriosis and psoriasis, are also associated with inflammation and neovascularization [40,44–46]. This phenomenon of nonspecific accumulation of macromolecules is referred to as the enhanced permeability and retention (EPR) effect and has been shown to work effectively for drug targeting through use of nanoparticulate formulations of drugs [47]. The requirements for probes employed to investigate the EPR effect are a high molecular weight and a relatively long circulation time to allow sufficient accumulation at these sites. By taking advantage of the EPR effect, iron oxide nanoparticles have been used in MRI for enhanced tumor visualization [48], for the visualization of arthritis [49] and for the investigation of the blood–brain barrier integrity after stroke [50], while intravenous administration of QDs has been shown to allow tumor visualization using fluorescence imaging [51]. Agents may also be directed to diseased sites by specific targeting of cell-surface receptors, either expressed at the vasculature or extravascular at sites with enhanced blood vessel permeability [52]. This is usually realized by conjugating an imaging probe with one or several targeting ligands, molecules that recognize and bind such receptors. Tumor angiogenesis can be visualized by targeting the αvβ3-integrin with probes for MRI [37,53,54], PET [55,56], SPECT [57], ultrasound [58] and fluorescence imaging [59]. Sipkins 310


and colleagues published an early report on the target-specific MRI visualization of tumor angiogenesis using paramagnetic polymerized liposomes conjugated with αvβ3-specifc antibodies [53]. The αvβ3-specifc arginine–glycine–aspartic acid (RGD) peptide has been used to target PET probes [55,56], QDs [29,59], iron oxide nanoparticles and bimodal liposomes [37] to this integrin. Perfluorocarbon microemulsions equipped with a RGD mimetic have been employed for angiogenesis visualization in tumor and atherosclerosis models [54,60]. Furthermore, these latter nanoparticles have been made specific for other imaging modalities, such as nuclear imaging [61], ultrasound [62] and optical techniques [61,63]. A variety of different cell-surface receptors, such as E-selectin, vascular cell adhesion molecule (VCAM)-1 and human epidermal growth factor receptor 2, have been visualized with targeted MRI [64–66] or optical probes [67]. A different strategy that may be pursued is the visualization of cells of the immune system that infiltrate and gather at the diseased sites. Labeling of such cells can be performed ex vivo and, after re-injection, these ‘visible’ cells can be tracked in vivo. Such cell-tracking studies performed with MRI are numerous in the fields of brain inflammation [12], stroke [68] and myocardial infarction [69], but have also been performed for the visualization of stem cells and the migration of T lymphocytes into tumors [70]. Although the tagged cells were successfully tracked in these studies, consideration of only the MRI signal changes is not always a reliable measure for cell infiltration and, importantly, cell viability. Labeled cells may excrete their content or excreted labels may be taken up by other cell types, which will result in a misinterpretation. Multiple labeling strategies, in which cells express green fluorescent protein, for example, and where the MRI probe is also labeled with a fluorescent marker to allow optical tracking, give a more thorough understanding of the MRI results [71–73]. This stresses the importance of multimodality investigations when conducting such studies. QDs have also been shown to have utility as a cell-tracking material. Tumor cells labeled with QDs have been administered intravenously in order to follow their extravasation into lung tissue. A second strategy used for cellular imaging is the intravenous administration of the probe, which associates with the cells either by active targeting or by a nonspecific uptake of the probe by the cells of interest. This approach future science group


has been used for monocyte tracking into inflamed sites and atherosclerotic plaques, although the exact mechanism of cell association is not yet fully understood [12]. The expression of enzymes or other molecules can also be visualized by so-called activatable probes that, upon interaction with their target, undergo a conformational change, aggregate or are processed by a sequence-specific enzyme [74]. An example of such a MRI agent is EgadMe [75]. This is a complex that contains a sugar moiety that prevents the water coordination to Gd3+ that is necessary for the complex to have high relaxivity. Enzymatic cleavage of this sugar by β-galactosidase improves the accessibility of water to Gd3+, which results in a considerable increase in the molar relaxivity. QD activation strategies have also been explored. Medintz and colleagues have designed a QD protein conjugate that functions as a sugar sensor [76]. The conjugate can be used to monitor the competition between cyclodextrin (conjugated to an acceptor dye) and maltose in binding to receptor proteins attached to the QDs. Fluorescence resonance energy transfer quenching occurs when the electronic excitation energy of the excited QD is transferred to a nearby nonfluorescent acceptor dye. The dye can be competitively removed from the conjugate by maltose, which results in recovery of the QD fluorescence. Another approach that has been developed recently is the so-called ‘self-illuminating’ QD conjugates [77]. QDs are conjugated to a mutant of the bioluminescent protein Renilla reniformis luciferase and, when this protein binds its substrate coelenterazine, it emits blue light that excites and produces fluorescence from the bound QD. In addition to the above strategies that have been employed to visualize disease processes with macromolecular contrast agents, the utility of endogenous nanoparticles has been recognized recently. Lipoproteins naturally accumulate in atherosclerotic plaques and tumors and, therefore, labeling such nanoparticles with magnetic and fluorescent molecules allows their visualization with MRI and optical techniques, respectively [28,78]. Similar labeling approaches have been shown with other endogenous nanoparticles, such as viruses [27,79] and ferritin [80,81]. All the above may be designed and applied for serial MRI and optical imaging studies by devising nanoparticulate agents that contain both fluorescent and magnetic properties. In the next sections, such particles will be discussed thoroughly. future science group


Fluorescent & magnetic nanoparticles Iron oxide-based multimodal nanoparticles Iron oxide nanoparticles, such as ultrasmall superparamagnetic iron oxide (50 nm) and superparamagnetic iron oxide (50–500 nm), are superparamagnetic contrast agents that cause negative contrast effects on T2*-weighted MR images [12], that is, they lead to a decreased MRI signal. This often makes the detection of the contrast difficult but, recently, techniques for ‘bright spot’, positive marker imaging of ultrasmall superparamagnetic iron oxides have been reported that facilitate the greater ease of detection [82–84]. Dextran cross-linked iron oxide particles (CLIOs) (Figure 1A) [85], equipped with fluorescent dyes [24], have been used in a variety of studies. These dual-modality imaging nanoparticles can be employed as agents that passively accumulate at target sites and have been shown to be useful for preoperative MRI and intraoperative optical imaging in an experimental brain tumor model [86], and also for the investigation of pancreatic inflammation in a model of diabetes [87]. CLIOs have been employed for multimodality imaging of atherosclerotic plaques [88] and have been used to show that monocyte accumulation in atheroma correlates with the extent of disease [89]. Conjugation of the cell-penetrating TAT-peptide to CLIO particles gives an agent that is very effective for labeling in vitro cells that are to be used in cell-tracking studies [90]. CLIOs cofunctionalized with NIR fluorescent dyes have been employed for combining noninvasive MRI with NIR fluorescent imaging [91]. In addition, a variety of ligands for targeting different markers of disease have been conjugated to CLIOs. In apoE-knockout mice, ex vivo imaging with VCAM-1-targeted nanoparticles revealed a good correlation between MR images and fluorescence imaging [92]. At 24 h, after adequate circulatory clearance of the particles, in vivo MR images revealed CLIO at VCAM-1-expressing plaques. In mouse models of cancer, CLIO particles have been used to target the αvβ3-integrin [93] and E-selectin [94]. An advanced application on the use of CLIOs was reported by Sosnovik and colleagues [95]. They used Cy5.5-labeled CLIOs conjugated with Annexin A5, a protein that binds the apoptosis-specific marker phospholipid phosphatidylserine, and used this nanoparticle for the multimodality visualization of apoptosis in infarcted mouse hearts in vivo. The challenge in 311


Figure 1. Cross-linked iron oxide for visualization of apoptosis in the mouse heart.

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(A) Schematic of the cross-linked iron oxide (CLIO) nanoparticles consisting of an iron oxide core (red sphere) covered with a cross-linked dextran coating (blue spheres). Targeting molecules or fluorescent dyes can be covalently linked to this particle (yellow spheres). (B & C) Cardiac magnetic resonance image of infarcted mice after the intravenous administration of (B) Annexin A5 Cy5.5–CLIO or (C) Cy5.5–CLIO. (D & E) Nearinfrared imaging reveals increased uptake of Annexin A5 C5.5–CLIO, compared with Cy5.5–CLIOs in the infarcted hearts. Reproduced with permission from [95].

this study was to generate high-quality and quantitative MR images of the beating mouse heart to allow quantitative differentiation between the heart walls of mice injected with a nonspecific and an apoptosis-specific agent. Infarction sites in the hearts of animals injected with Annexin A5 CLIOs had a significantly lower T2* than infarction areas of the heart of animals injected with nonspecific CLIOs (Figure 1B & C). In addition, ex vivo NIR fluorescence imaging of excised hearts showed a good correlation with the in vivo MRI findings (Figure 1D & E). CLIO nanoparticles also have potential as activatable probes. Fluorescent quenching of an attached fluorochrome can occur in part due to an interaction with iron oxide when the payload of fluorescent dyes is low (less than one dye molecule per particle) [24]. If the dye is conjugated to CLIO particles in such a fashion that it can be cleaved by an enzyme, the fluorescence of the dye will be restored after dissociation from the particle. These probes may then provide the basis for a new class of so-called smart nanoparticles, whose position may be pinpointed through their magnetic properties, while providing information on their molecular environment by optical imaging techniques. Recently, an iron oxide-based polymeric nanoparticle has been reported that has multiple functional units to allow vascular targeting, photodynamic therapy and imaging with MRI and optical techniques [96]. The nanoparticle 312


(Figure 2A & B) consisted of polyacrylamide that was embedded with iron oxide, fluorochromes and a photodynamic dye, while F3 peptides at the end of poly(ethylene glycol) (PEG) chains were attached to target these nanoparticles to cancer cells. This agent was administered to rats with intracranial 9L gliomas, for which the authors demonstrated efficient targeting in vitro, as evidenced with fluorescence microscopy (Figure 2C). In vivo targeting to brain tumors was monitored with MRI (Figure 2D & E) and the photodynamically induced therapeutic effect was evaluated with diffusion MRI (Figure 2F).

Iron oxide-containing micelles

Nanoparticles, such as iron oxide particles or QDs, are mostly synthesized in nonpolar organic solvents. If they are to be solubilized in aqueous buffers, their hydrophobic surface ligands must be replaced by amphiphilic ones. An alternative strategy is incorporating these hydrophobic particles into the core of micelles formed from amphiphilic block co-polymers or PEGylated lipids [97,98]. This elegantly simple procedure allows the solubilization of different nanosized hydrophobic materials and may also be employed to create multifunctional particles of small size. Functionalization of such nanoparticles can be easily realized through use of the appropriate block co-polymer and subsequent reaction. Furthermore, fluorescent properties can be introduced by the inclusion of fluorescent dyes, either as an future science group


Figure 2. Polyacrylamide nanoparticles for the imaging and therapy of brain tumors. Photosensitizer/ photodynamic agents F3 targeting Magnetic/ moiety contrast D agent A

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agent might be very useful for detecting apoptotic cells in pathological processes such as ischemia reperfusion injury, atherosclerosis and tumor treatment. Prior to this report, Nitin and colleagues used a similar approach to solubilize iron oxide [98]. They conjugated TAT peptides and a fluorescent label to the distal end of the PEG chains of the phospholipids to coat the iron oxide particles. The TAT peptide has been shown to deliver nanoparticles into cells, making it attractive for intracellular delivery. The uptake of this conjugate was demonstrated in vitro with both MRI and fluorescence microscopy. In addition to PEG lipids, amphiphilic block copolymers have also been used to solubilize iron oxide and to create multifunctional nanoparticles. Careful control of the preparation parameters allows a defined inclusion of one or multiple magnetite nanoparticles per micelle [100]. Interestingly, mixtures of hydrophobic nanomaterials can also be included in the core of micelles. This approach has been used to create micelles that contained doxorubicin, a cytostatic agent with fluorescent properties, and iron oxide. Subsequent conjugation of RGD peptides allowed the specific targeting and treatment of tumor endothelial cells in vitro [101]. Paramagnetic & fluorescent liposomes

(A) Depiction of the contrast agent. The nanoparticle was formed from polyacrylamide, which was embedded with photodynamic dyes (Photofrin®) and imaging agents. Poly(ethylene glycol) linkers allowed the conjugation of a targeting peptide. (B) Scanning electron microscopy image of the nanoparticles. (C) Targeted particles associated with endothelial cells in vitro, as evidenced by fluorescence microscopy. (D) T2-weighted magnetic resonance image of a brain tumor in a rat. (E) The particle accumulation in the tumor was also visualized using T2*-weighted magnetic resonance imaging. (F) Diffusion map after photodynamic therapy. Reproduced with permission from [96].

amphiphile or by covalent linkage to the micelle surface. An example of this exciting new imaging platform are PEG lipid-based micellar iron oxide nanoparticles functionalized with Annexin A5 (Figure 3A) that were used for the detection of apoptotic cells [99]. In vitro, these Annexin A5-conjugated particles showed high affinity for apoptotic cells, which resulted in a large decrease of T2 of a pellet of these cells and a darkening of the images of the cell pellet (Figure 3B). The inclusion of a fluorescent amphiphile allowed fluorescence microscopy of apoptosis (Figure 3C). In vivo, this contrast future science group


Liposomes are spherical, self-closed structures formed by one or several concentric lipid bilayers with an aqueous phase in the core and between the lipid bilayers. They have been exploited for drug-delivery purposes for several decades [102] and their application as carriers of MRI contrast agents was first reported in the 1980s [3]. There are two approaches for paramagnetic material delivery using liposomes. In the first approach, the MRI agent is included in the aqueous interior. Recently, liposomes that include gadodiamide and doxorubicin via this method were used to study the liposomal distribution after convection-enhanced delivery to brain tumors in rats and mice [103]. In a second class of liposomal contrast agents, the paramagnetic molecules are incorporated into the lipid bilayer, which makes the amphiphilic paramagnetic complexes an integral part of the liposomal surface. This approach results in an improved ionic relaxivity of the metal compared with the approach of encapsulating the paramagnetic molecules in the aqueous interior, thus resulting in a better contrast agent [3]. 313


Figure 3. Imaging apoptosis with micellar iron oxide. A

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(A) Schematic representation of the micellar iron oxide that contained fluorescent amphiphiles (green) and was functionalized with the apoptosisspecific protein Annexin A5 (red). (B) The uptake of the contrast agent resulted in a signal decrease of the cell pellet of cells that were incubated with Annexin A5-functionalized micellar iron oxide (compare cell pellets). (C) The specific binding of the agent with apoptotic cells was visualized with fluorescence microscopy.

Polymerized paramagnetic liposomes were introduced in 1995 by Storrs and colleagues [9]. The diameter of these particles was 300–350 nm and they contained a 30% payload of gadolinium, making them very effective. This agent was functionalized with αvβ3-specific antibodies to allow the detection of angiogenesis in tumorbearing rabbits [53] and to detect the expression of leukocyte-adhesion molecules in the brain of a mouse model of multiple sclerosis [104]. Mulder and colleagues introduced a PEGylated liposomal contrast agent for dualmodality imaging of molecular markers with both MRI and fluorescent techniques (Figure 4A) [11]. This contrast agent has been made target specific for E-selectin with monocolonal antibodies [11], for apoptosis with Annexin A5 proteins [99] and for angiogenesis with αvβ3-specifc RGD peptides [37]. This nanoparticulate contrast agent conjugated with cyclic RGD peptides was used to identify the angiogenic endothelium in tumor-bearing mice with in vivo MRI and ex vivo fluorescence microscopy. The cyclic RGD peptide has high affinity for the αvβ3-integrin, which is upregulated at endothelial cells of angiogenic blood vessels. MRI revealed that, upon intravenous injection of the contrast agent, the RGD liposomes localized to a large extent in the tumor rim, which is known to have the highest angiogenic activity (Figure 4B). The specific and 314


exclusive targeting of this agent to tumor blood vessels was confirmed by ex vivo immunofluorescence (Figure 4C). This study demonstrated the importance of validating the MRI findings with a complementary technique, such as fluorescence microscopy. In addition to angiogenesis visualization, this approach was also employed to quantify angiogenesis and to evaluate the effects of angiostatic therapy with MRI in vivo [105]. Mice subcutaneously inoculated with B16F10 melanoma cells were treated either 3 or 14 days later with the angiogenesis inhibitors anginex or endostatin. The percentage of the tumor volume with significant signal enhancement upon intravenous injection of αvβ3-specific liposomes was quantified for each case. Subsequent to the MRI measurements, microvessel density (MVD), which is an ex vivo surrogate marker for angiogenic activity, was determined. It was found that in vivo molecular MRI analysis of tumor angiogenesis closely reflected the treatment effects, as deduced from ex vivo MVD determinations (Figure 4D) and therefore may serve as a useful surrogate marker of angiogenic activity. Micelles for target-specific dual-modality imaging

Micelles are amphiphilic aggregates that are particular interesting in drug-delivery applications for carrying poorly soluble pharmaceutical agents [106]. Ligands can also be coupled to the surface of the micelles to target them to specific sites. These properties also make micelles excellent candidates for target-specific MRI. The most common strategy for the preparation of micelles with paramagnetic properties is to use mixtures of amphiphilic molecules, some of which include a Gd3+-chelating and hydrophilic headgroup and some amphiphiles that strongly promote micelle formation. Tween micelles composed of phospholipids, a surfactant, an amphiphilic fluorophore and an amphiphilic gadolinium contrast agent were recently used for detecting macrophages in ApoE-knockout mice using MRI in vivo and using immunofluorescence [25,26]. Macrophages are known to play a central role in the pathogenesis and evolution of atherosclerotic plaques. The contrast agent was targeted to macrophages in atherosclerotic plaques by coupling biotinylated antibodies, specific for the macrophage scavenger receptor, to the micelles via an avidin–biotin linkage. A signal increase of the vessel wall at 1 and 24 h postinjection compared with precontrast imaging was observed with MRI, future science group


Figure 4. Quantitative molecular imaging of tumor vasculature with RGD liposomes.

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(A) Schematic of the contrast agent. The liposomes are composed of carrier lipids (yellow), fluorescent lipids (green) and paramagnetic amphiphiles (purple) and are conjugated with RGD peptides (green pentagons). (B) Magnetic resonance images of a slice through the tumor of an animal that was injected with paramagnetic RGD liposomes. T1-weighted image measured after the injection of the RGD-conjugated liposomes revealed a signal increase of the tumor rim (yellow arrow). Pixels in the tumor with signal enhancement of at least three times the noise level are color coded according to the pseudocolor scale on the right. (C) Fluorescence microscopy of DAPI-colored 10-µm sections from dissected tumors of mice that were injected with RGD liposomes. Comparison of histologically determined microvessel density (D) (i) with significantly enhanced tumor area using αvβ3-targeted molecular magnetic resonance imaging (D) (ii). The mice received angiogenic treatment with anginex and endostatin for 3 or 14 days, or were left untreated (control).

which corresponded with anatomically matched areas of histologically determined macrophagerich plaques (Figure 5A). Fluorescence microscopy revealed the specific association of the targeted micelles with macrophages (Figure 5B). Perfluorocarbon nanoparticles

A nanoparticulate imaging platform that has been exploited for most of the available imaging methods is perfluorocarbon microemulsions [107]. These nanoparticles have a perfluorcarbon core that is covered with a monolayer of lipids. This lipid layer can be used to include payloads of contrast agents and drugs and to attach targeting ligands. These nanoparticles have been used as molecular imaging agents for ultrasound [62], MRI [108], nuclear techniques [61] and computed tomography [109]. In addition, immunofluorescence and in vitro microscopy studies have been performed by fluorescently

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labeling the nanoparticles [61,63]. Recently, fluorine MRI was performed on these nanoparticles, which allowed their detection in a so-called hotspot fashion, without any background [33,34]. While 1H MRI can be used to obtain detailed anatomical information, 19F NMR spectroscopy makes precise nanoparticle quantification and the detection of multiple agents possible. A variety of molecular markers have been visualized using perfluorocarbon nanoparticles, including fibrin [108], αvβ3-integrin [54,60,110] and tissue factor [10]. Incorporation of high payloads of paramagnetic lipids (Gd–diethylene-triamine-pentaacetic-acid-2-benzoxazolinone [Gd–DTPA–BOA]) in the lipid monolayer makes this a high relaxivity nanoparticle suitable for the detection of sparse epitopes with MRI. Perfluoronanoparticles targeted to the αvβ3-integrin have been used for detecting angiogenesis in tumor and atherosclerosis models

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Figure 5. Macrophage imaging in atherosclerosis using immunomicelles.

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(A) (i–iii) In vivo magnetic resonance images obtained at baseline and postinjection of macrophage-targeted immunomicelles in ApoE-knockout mouse. The magnetic resonance image insets are enlargements of the abdominal aortas. (iv) Hematoxylin and eosin section of the aorta at the identical anatomic level as the MRI image from the same animal. (B) Confocal laser-scanning microscopy image demonstrating colocalization of fluorescently labeled (C6-7-nitro-2,1,3-benzoxadiazol-4-yl) immunomicelles (green) and anti-CD68-stained macrophages (red). Cell nuclei were stained with DAPI (blue). [54,60,111]. In Figure 6A–C, MRI images of a tumor-bearing mouse before and after the administration of αvβ3-specific perfluorocarbon nanoparticles are depicted. Fluorescently labeled versions of these nanoparticles injected into a Vx-2 tumor in a rabbit were shown to associate with the tumor vasculature through histology (Figure 6D) and immunofluorescence (Figure 6E). Atherosclerotic plaques contain angiogenic microvessels, which are believed to play an important role in the plaque development. Imaging plaques and tumors with an αvβ3-specific contrast agent, therefore, is of importance for early detection, defining the severity of the disease and following the effect of therapy. Winter and colleagues published results on a combinatory approach of MR molecular imaging and drug targeting of atherosclerosis with this approach [112]. To that end, they used the αvβ3-specific nanoparticles to target the aortic vessel wall of atherosclerotic rabbits. For therapeutic purposes, they included fumagillin in the lipid monolayer of the nanoparticles and observed an anti-angiogenic effect with MRI that was confirmed histologically.

Dendrimers

Dendrimers are synthetic macromolecules comprised of branched repeat units that emanate radially from a point-like core [113]. Functional groups are usually situated on the dendrimer surface and these groups may be exploited for multiple purposes, such as the conjugation of Gd

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chelates, fluorophores or targeting molecules. The number of branches determines both the size of the particle and the quantity of functional moieties that can be introduced. This allows, in contradiction to supramolecular assemblies such as liposomes and micelles, the creation of particles of completely defined size and composition. Several studies have shown the applicability of dendrimers as macromolecular MRI contrast agents, for example in the MRI-based evaluation of tumor permeability [114–118]. Talanov and colleagues have used generationsix dendrimers to create a dual-modality contrast agent [119]. To that end, they conjugated Gd chelates and a NIR dye to the surface of the dendrimers and demonstrated their application for visualization of sentinel lymph nodes in mice with both MRI and fluorescence imaging. Endogenous nanoparticles

Endogenous nanoparticles, such as lipoproteins and viruses, have also been modified for use as contrast agents [27,28,78,120]. For example, Gd-loaded apoferritin has been shown to function as a high relaxivity contrast agent [80,81]. The ferritin protein complex consists of 24 protein subunits, and the procedure for loading this nanoparticle with Gd chelates is elegant but simple [81]. At low pH, the subunits are present as monomers, which allows their mixing with Gd chelates. Upon increasing the pH to physiological values, the subunits reform the complex and the Gd chelates are trapped and compartmentalized



Figure 6. Molecular imaging angiogenesis using αvβ3-specific perfluorocarbon nanoparticles.

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(A–C) T1-weighted magnetic resonance image of a mouse (A) before and (B,C) 1 h after the injection of αvβ3-specific paramagnetic perfluorocarbon nanoparticles. (D) Light microscopy image of Vx-2 adenocarcinoma and capsule. (E) Fluorescent microscopy image of tumor capsule region depicted in (D). The green signature of vessels retaining αvβ3-integrin-targeted nanoparticles (green) within the capsule (yellow arrows). DAPI was used to stain nuclei. Reproduced with permission from [61,111].

within the ferritin cavity. This nanoparticle can be conjugated with targeting ligands and employed as a target-specific agent. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) play important roles in the transport of cholesterol. LDL consists of a lipid core of cholesterol esters and triglycerides covered by a phospholipid monolayer that contains a large apolipoprotein. LDL binds to the LDL receptor exclusively via this apoliporotein and is subsequently internalized. The overexpression of the LDL receptor is associated with several pathologies, including atherosclerosis and cancer. Corbin and colleagues have used LDL enriched with Gd chelates and fluorescent dyes to specifically visualize tumors that overexpress LDL receptors [78]. A HDL-based particle was developed by Frias and colleagues for dual-modality imaging [28,121]. They constituted the particle from HDL future science group


apolipoproteins and phospholipids, with or without unesterified cholesterol. A paramagnetic phospholipid, Gd–DTPA–DMPE, and a fluorescent phospholipid were incorporated (Figure 7A) for imaging purposes. This contrast agent was applied to a mouse model of atherosclerosis and a succession of in vivo MRI images was taken. The vessel wall of the abdominal aorta showed strong signal enhancement with a maximum 24 h post-injection (Figure 7B). Following aorta excision, it was established with confocal fluorescence microscopy on histological coupes that the contrast agent was associated with macrophages (Figure 7C). The lipoprotein contrast agent platform may be of great use for the noninvasive characterization of atherosclerosis and cancer. Recently, two studies concerning the use of viruses for contrast-enhanced MRI have appeared in the literature. In the first approach, the protein cage of Cowpea chlorotic mottle virus was modified [79]. This virus has 180 metal-binding sites that were used to complex Gd3+. Another approach was developed by Anderson and colleagues [27]. They conjugated more than 500 Gd chelate groups onto a viral capsid. To allow complementary detection with optical techniques, conjugation of fluorescein was realized in a similar fashion. QDs for dual-modality imaging

Semiconductor nanocrystals (QDs) have unique optical properties that make them ideally suited for a number of applications in biomedical imaging, with several important advantages over fluorescent dye molecules [76]. The absorption spectrum of a QD is characterized by a very broad band, since any photon with energy equal to or higher than the band-gap is absorbed, while the emission spectrum is rather narrow (full widths at half maximum are in the 25–40-nm range). The optical properties of QDs can be tuned by judicious control of composition and size, allowing emission wavelengths spanning from the near-UV to NIR to be obtained, making QDs particularly suitable for multiplexed imaging. Moreover, the surface of QDs can be easily modified so that new functionalities and properties can be introduced. However, QDs are potentially cytotoxic if allowed to interact with the cellular environment and are prone to photochemical degradation, albeit to a lesser extent than dyes. These shortcomings have been (partially) overcome by the use of core–shell QDs and/or suitable coatings. 317


Figure 7. Paramagnetic and fluorescent high-density lipoprotein for atherosclerotic plaque visualization.

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(A) High-density lipoprotein-based nanoparticle enriched with a fluorescent amphiphile (DPPE–NBD) and a paramagnetic amphiphile (Gd–DTPA–DMPE). (B) In vivo magnetic resonance imaging at the level of the abdominal aorta as measured before and 1-, 24- and 48-h postinjection. (C) Light microscopy of a histological section. (D) The arrow indicates the association of the high-density lipoprotein-based nanoparticle with macrophages, as revealed by fluorescence microscopy. DMPE: Dimyristoyl phosphatidylethanolamine; DPPE: Dipalmitoyl phosphatidylethanolamine; DTPA: Diethylene-triamine penta-acetic acid; NBD: N-(7-nitro-2-1,3-benzoxadiazol-4-yl).

Dubertret and colleagues introduced QDs with an amphiphilic coating for in vivo imaging [97]. Water-insoluble QDs are usually dissolved in apolar solvents, such as toluene or chloroform. The hydrophobicity of the QDs is caused by hydrophobic capping molecules. A micellar coating can subsequently be applied by adding an excess of PEGylated lipids to a QD–chloroform solution, evaporating the solvents to form a mixed film of PEG lipids and QDs and hydrating and heating this film to induce the formation of micelles that contain a QD in their core. 318


Mulder and colleagues extended this procedure by the incorporation of a paramagnetic amphiphile in the micellar shell to allow parallel detection of this nanoparticle by both optical methods and MRI [29]. Furthermore, the PEG chains of the micelles can be functionalized with targeting ligands. In Figure 8, a schematic of a RGD peptide-conjugated paramagnetic QD (pQD)-micelle is depicted. The ionic relaxivity r1 of the QD–micelle at a clinically relevant field strength of 1.41 T was more than 12 (mMs)-1, which is three times higher than that of future science group


Figure 8. Quantum dot micelles for multimodality imaging of angiogenesis. Probe A

RGD peptide

Paramagnetic lipid PEGylated lipid Quantum dot

B

In vivo tumor vasculature imaging IVM

MRI

(A) Schematic representation of a quantum dot–micelle conjugated with RGD peptides. (B) IVM revealed tumor vasculature labeling at the cellular level (arrows indicate the endothelium), while MRI was used to visualize tumor vessels at the anatomical level. IVM: Intravital microscopy; MRI: Magnetic resonance imaging; RGD: Arginine–glycine–aspartic acid.

Gd–DTPA at this field strength. Since the QD–micelles contain approximately 300 lipid molecules, half of which are Gd–DTPA–BSA, the relaxivity per pQD is estimated to be approximately 2000 (mMs)-1. This high relaxivity makes the pQD contrast agent an attractive candidate for molecular MRI purposes. The QD-based contrast agent was used to carry out molecular imaging of tumor angiogenesis at the cellular and anatomical level. Tumor-bearing mice were intravenously injected with the contrast agent and were studied with either intravital microscopy or MRI in vivo. Intravital microscopy was used for the real-time monitoring of the fate of injected QDs. In Figure 8B, a fluorescence image of blood vessels of a tumor is depicted 30 min after the injection of contrast agent. High-resolution T1-weighted MRI was performed before and after applying the agent. Figure 8C shows a colorcoded image generated from the MRI data future science group


before and 45 min after contrast agent injection. The MR imaging voxels that showed significant signal enhancement following RGDconjugated QD–micelle injection were mainly found at the tumor periphery, which corresponds with the regions of the tumor with highest angiogenic activity. Apoptosis, or programmed cell death, has also been investigated using this QD-based probe [122]. To that end, the QD–micelles were conjugated with Annexin A5, a protein that binds specifically to phospatidylserine, a phospolipid expressed at the outer lipid layer of apoptotic cells, as mentioned previously. Recently, a similar approach for the visualization of apoptosis has been developed where commercially available streptavidin-coated QDs were functionalized with biotinylated Annexin A5 and paramagnetic dendritic wedges [123]. Using this QD-based conjugate, apoptosis could be detected with MRI and optical techniques in vitro. Conclusion In this article, we have reviewed multimodality nanoparticles that have both magnetic properties for MRI and fluorescent properties for optical imaging. MRI has proven to be a very versatile and powerful diagnostic tool in the clinical and research setting. Contrast agents for MRI are becoming more specific and more effective in terms of their relaxivity. This permits the utilization of MRI for molecular imaging purposes. Optical techniques are progressing rapidly for their employment in vivo, but are limited by the tissue penetration of light. However, they allow a fast and sensitive evaluation of fluorescent probes, while generating little background signal. The aforementioned techniques are highly complementary and their combination has significant advantages, especially in the field of molecular imaging. We have shown that there has been considerable progress in the development of nanoparticulate probes for dual-modality imaging and that their application greatly improves the in vivo visualization of pathophysiology at the tissue, cellular and molecular level in diseases such as cancer, atherosclerosis, myocardial infarction, stroke and diabetes. Future perspective There is an ongoing effort in the development of novel nanoparticles that incorporate multiple properties. In addition to the imaging techniques that are the focus of this review, these nanoparticles may be designed to allow their detection 319


with other modalities, such as computed tomography. Therapeutic properties may be implemented to further extend this platform and to allow the visualization of the intervention. Importantly, progress in imaging techniques in the molecular biology that underlies the pathology as well as in the search for target-specific molecules will broaden the use of multimodality nanoparticles. A

major future challenge is translation of the technology to the clinic. This will require a great effort to optimize the probes in terms of their immunogenicity, toxicity, their specificity and interference with biological processes and their accumulation in the liver and spleen. Finally, the demonstration of efficacy of the new contrast agent compared with existing methods must be shown.

Executive summary • Magnetic resonance imaging (MRI) and optical imaging methods are highly complementary techniques in terms of their ability to image opaque tissue, their resolution, sensitivity, anatomical definition and in terms of methods to generate contrast. Therefore, combining these techniques has significant advantages in the field of experimental and molecular imaging. • MRI is capable of generating 3D images of opaque tissue with high spatial resolution and is available in both experimental and research settings. • Optical techniques use fluorescence to distinguish multiple probes simultaneously using microscope or whole-body imaging. • Developments in nanotechnology offer the possibility of creating various types of nanoparticles that have multiple integrated properties. • Fluorescent and magnetic nanoparticles can be created from a wide range of materials and molecules, including iron oxide, quantum dots, amphiphilic molecules or even endogenous lipoproteins. • Reactions may be performed on nanoparticle surfaces to allow the inclusion of multiple properties, such as poly(ethylene glycol) (PEG)ylation for improved circulation time, the addition of magnetic and/or fluorescent moieties or the conjugation of targeting ligands. • Fluorescent and magnetic diagnostic nanoparticles can be employed to visualize vascular permeability, the expression of cell-surface receptors and to track certain cell types. • Experimental models of cancer, atherosclerosis, myocardial infarction and pancreatic inflammation are among the pathologies already imaged using these combined technologies. There is enormous potential for imaging the progression and the underlying biochemistry of many other diseases using this multimodality approach.

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