Multimodal probes have been demonstrated to be useful when combined with labels for different, preferably complementary imaging modalities. One of the first examples of a bimodal probe was reported by Huber, Meade et al.32
They described bifunctional contrast-enhancing agents for optical and MRI. The main advantage of this combination is that it integrates optical properties for high-resolution fluorescence microscopy and imaging and magnetic properties for in vivo
MRI visualization of intact and opaque organisms. Various other fluorescently labeled MRI probes have also been explored, of which an overview can be found in two excellent review papers.33,34
In addition to fluorescently labeled MRI probes, many other types of dual imaging probes have also been developed. For example, Cai et al. developed a QD probe that was additionally labeled with 64
Cu to enable its visualization by PET imaging.35
More recently, nanoprobes that exhibit features for their visualization with more than two imaging modalities have been investigated. Paramagnetic QD-micelles () were introduced by Mulder et al. in 2006.36
In this study it was shown that QDs encapsulated in a monolayer of polyethylene glycol(PEG)ylated and paramagnetic lipids exhibit excellent features for both optical techniques and MRI. Moreover, the PEGylated lipids were functionalized with RGD-peptides to introduce specificity for the angiogenic marker α
3-integrin. In a follow-up study targeted multimodality imaging of tumor angiogenesis was performed on tumor-bearing mice that were intravenously injected with the α
3-specific QD-micelles and were studied with three complementary in vivo
Intravital fluorescence microscopy was used for the real-time monitoring of the fate of injected QD-micelles. In , a brightfield and a fluorescence image of tumor blood vessels are depicted 30 min after administration of the QD-micelles. Another optical technique, whole body fluorescence imaging, enabled the visualization of particle accumulation with high sensitivity and high temporal resolution in mice that had a tumor in their kidney (). As a third imaging modality, high-resolution T1-weighted MRI was performed before and 45 min following QD-micelle administration and revealed significant signal enhancement that was mainly found at the tumor periphery (), which corresponds with the regions of the tumor with highest angiogenic activity.
FIGURE 2 Paramagnetic QD-micelles for multimodality imaging. (a) Schematic depiction of αvβ3-specific and paramagnetic QD-micelles. (b) Intravital microscopy brightfield (left) and fluorescence (right) images of microvessels in tumor-bearing mice (more ...)
Iron oxide nanoparticles have shown great utility in the field of target-specific molecular MRI. Some pioneering studies were performed by Weissleder and colleagues who demonstrated the application of dextran coated iron oxide nanoparticles conjugated to human holo-transferrin to image transgene expression in tumor-bearing mice.38
In the field of cardiovascular disease a number of processes have been studied using iron oxide facilitated MR molecular imaging, including apoptosis after myocardial infarction39
and the upregulation of cell adhesion molecules in mouse models of atherosclerosis.40
In the aforementioned studies these iron oxide probes were additionally labeled with a fluorescent dye to allow colocalization with immunofluorescent techniques and perform near-infrared fluorescence (NIRF) imaging of live animals and excised organs, such as the heart and aorta. Most recently, Nahrendorf, Weissleder, and colleagues have shown two approaches to additionally label these magnetofluorescent probes with a radiolabel to allow their visualization with PET.41,42
In one approach the fluorescent and magnetic nanoprobes were conjugated with 18
F-based radiotracer via so-called ‘click’ chemistry,41
while in another approach the chelator diethylenetriaminepentaacetic acid (DTPA) was conjugated to the nanoparticle dextran coating to allow complexation with the radiotracer 64
The latter nanoparticle platform was employed to quantitatively study macrophage inflammation in the aorta of atherosclerotic apolipoprotein E knockout (apoE-KO) mice. In a CT and combined PET/CT image reveal enhancement of the posterior aortic root, an area of the aorta that is known to have a high plaque burden in this mouse model. MRI scans of the aortic root prior to () and postadministration () of the triply labeled nanoprobe revealed a decrease of T2, which is indicative of iron oxide accumulation. Moreover, NIRF imaging of excised and intact aortas revealed the distribution of the nanoprobe in the atherosclerotic aorta and confirmed a prominent accumulation in the aortic root ().
FIGURE 3 (a) Schematic of the trimodality reporter 64Cu-TNP. The chelator DTPA allows attachment of radiotracer 64Cu, the iron oxide core provides contrast in MRI and the fluorophore for fluorescence techniques. (b), (c) 64Cu-TNP accumulates in atherosclerotic (more ...)
Silica nanoparticles can be synthesized in a wide range of desired sizes (50–1000 nm) and in the past decade, silica-based nanoparticles have increasingly been exploited for biomedical applications, including drug and gene delivery as well as a carrier vehicle for different contrast-generating materials. Despite the intrinsic utility of silica for biomedical applications, a serious drawback of these inorganic nanoparticles is their inherently low biocompatibility. To address this issue Koole et al. have recently developed a novel method to obtain hydrophobic silica nanoparticles coated with a physically adsorbed monolayer of PEGylated phospholipids (, top).43
This highly flexible coating method allows, next to the inclusion of PEGylated lipids, the incorporation of many other lipid species, e.g., paramagnetic lipids for MRI and bio-functional lipids to achieve target specificity. In a recent study this nanoparticle platform was functionalized with α
3-specifc RGD-peptides and it was shown with fluorescence microscopy, MRI, and fluorescence imaging that these particles were preferentially taken up by proliferating endothelial cells (HUVEC, human umbilical vein endothelial cells) in vitro
. The cytotoxicity and pharmacokinetics of paramagnetic and PEG-lipid coated QD containing silica nanoparticles was studied by van Schooneveld et al. using a variety of imaging techniques.44
Lipid-coated silica exhibited a prolonged circulation half-life as determined by quantitative fluorescence imaging of blood samples and in vivo
MRI (). Confocal microscopy () and transmission electron microscopy (TEM) () of tissue sections of mice sacrificed 24 h after the administration of lipid-coated silica nanoparticles revealed the particles to accumulate in the liver and spleen. Interestingly, the bare silica particles were also found to accumulate in the lungs of the animals, indicating the value of the lipid coating.
FIGURE 4 Top: Schematic depiction of quantum dot containing silica particle with a lipid coating. (a) Determination of blood circulation half-life values by fluorescence imaging (left) and magnetic resonance angiography (right). (b) Confocal microscopy of sections (more ...)
A number of studies have focused on nanoparticulate MRI probes that were also labeled for optical detection. Interestingly, the nature of the majority of such particles allows their visualization with TEM, rendering them trimodal. Excellent examples based on silica nanoparticles were independently published by Kim et al.,45
Rieter et al.,46
and Zhang et al.47
The combination of using different imaging techniques, as demonstrated in the abovementioned studies, has shown to be very valuable for a variety of reasons. PET/CT imaging allows quantification of nanoparticle accumulation, be it with a relatively poor spatial resolution, while MRI methods can generate images of opaque tissue with a very good spatial resolution, superb tissue contrast, albeit with limited sensitivity to detect contrast-generating materials. Optical techniques have the advantage of a high sensitivity and the capability to detect multiple species simultaneously, but the penetration depth of light is limited which restricts the application of these techniques to the surface or small species. Microscopic techniques allow the investigation of the (sub)cellular distribution of nanoparticulate materials, while electron microscopy techniques can visualize cellular organelles and can also identify individual particles within these organelles. Therefore, these combinatory studies allow investigators to study particle distribution, kinetics, and fate at different levels, ranging from the entire organism down to cellular and particle level.
Recently, Cormode et al. modified high density lipoprotein (HDL) particles to create endogenous nanoparticle-inorganic material composites.48
A method was developed to modify both the hydrophobic core and the phospholipid coating to provide contrast for medical imaging. A variety of nanocrystals, i.e., gold nanoparticles, iron oxide nanoparticles, and QDs, were included in the HDL core to produce a broad range of novel contrast agents for multimodality imaging. The gold core HDL was additionally labeled with fluorescent and paramagnetic lipids to create a trimodal particle that has properties for detection with CT, fluorescence, and MRI (). Characterization of the particles revealed these nanoparticles to be very similar to native HDL.
FIGURE 5 (a) Schematic depiction of gold core high density lipoprotein (Au-HDL). The particle corona consists of ordinary phospolipids, paramagnetic lipids, fluorescent lipids and apolipoprotein A-I. T1-weighted MR images of the aorta of an apoE-KO mouse (b) before (more ...)
In vitro experiments revealed that these particles were preferentially taken up by macrophages, as evidenced by confocal laser scanning microscopy, cell pellet MR imaging, CT imaging, and TEM. In vivo experiments using the gold core HDL nanoparticles were performed with the apoE-KO mouse model of atherosclerosis. The abdominal aorta of mice injected with Au-HDL appeared brighter on T1-weighted MR images at 24 h (, respectively), while ex vivo confocal microscopy of aortic sections that were stained for macrophages (green) and for nuclei (blue) revealed the Au-HDL particles (red) to be associated with macrophages (). CT images of intact aorta specimen from Au-HDL-injected and control animals revealed specific uptake of Au-HDL (). Importantly, these CT data were corroborated with fluorescence imaging of the same specimen ().