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Purpose: This study was aimed to assess the effects of High Intensity Focused Ultrasound (HIFU) thermal ablation on tumor T1ρ ., Methods: In vivo T1ρ measurements of murine tumors at various spin-lock amplitudes (B1 = 0-2000 Hz) were performed before (n = 13), directly after (n = 13) and 3 days (n = 7) after HIFU treatment. T2 maps were obtained from the measurements at B1 = 0 Hz., Results: Average tumor T1ρ distributions at the different experimental time points showed a shift toward lower T1ρ values after HIFU for all spin-lock amplitudes, which became larger with increasing spin-lock amplitude at 3 days after treatment. Statistical analysis confirmed a significant effect of spin-lock amplitude on the average change in T1ρ (ΔT1ρ ) as compared to baseline at 3 days after HIFU. At 3 days after treatment, ΔT1ρ values at B1 above 100 Hz were significantly lower (more negative) than at B1 = 0 Hz (T2 )., Conclusion: Significant changes in tumor T1ρ were observed after HIFU treatment. These T1ρ changes were distinctly more pronounced than HIFU-induced changes in T2 . The results indicate that T1ρ imaging is sensitive to HIFU-induced tissue changes and may thus be a suitable MR method for the evaluation of HIFU treatment. Magn Reson Med 73:1593-1601, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E deficient mice ( Apoe -/- ) with advanced atherosclerotic plaques. This resulted in rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an eight-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

Purpose: To develop a protocol for diffusion-tensor imaging (DTI) of the complete upper legs and to demonstrate feasibility of detection of subclinical sports-related muscle changes in athletes after strenuous exercise, which remain undetected by using conventional T2-weighted magnetic resonance (MR) imaging with fat suppression., Materials and Methods: The research was approved by the institutional ethics committee review board, and the volunteers provided written consent before the study. Five male amateur long-distance runners underwent an MR examination (DTI, T1-weighted MR imaging, and T2-weighted MR imaging with fat suppression) of both upper legs 1 week before, 2 days after, and 3 weeks after they participated in a marathon. The tensor eigenvalues (λ1, λ2, and λ3), the mean diffusivity, and the fractional anisotropy (FA) were derived from the DTI data. Data per muscle from the three time-points were compared by using a two-way mixed-design analysis of variance with a Bonferroni posthoc test., Results: The DTI protocol allowed imaging of both complete upper legs with adequate signal-to-noise ratio and within a 20-minute imaging time. After the marathon, T2-weighted MR imaging revealed grade 1 muscle strains in nine of the 180 investigated muscles. The three eigenvalues, mean diffusivity, and FA were significantly increased (P < .05) in the biceps femoris muscle 2 days after running. Mean diffusivity and eigenvalues λ1 and λ2 were significantly (P < .05) increased in the semitendinosus and gracilis muscles 2 days after the marathon., Conclusion: A feasible method for DTI measurements of the upper legs was developed that fully included frequently injured muscles, such as hamstrings, in one single imaging session. This study also revealed changes in DTI parameters that over time were not revealed by qualitative T2-weighted MR imaging with fat suppression., (© RSNA, 2014.)

We introduce a fast protocol for ultra-short echo time (UTE) Cine magnetic resonance imaging (MRI) of the beating murine heart. The sequence involves a self-gated UTE with golden-angle radial acquisition and compressed sensing reconstruction. The self-gated acquisition is performed asynchronously with the heartbeat, resulting in a randomly undersampled kt-space that facilitates compressed sensing reconstruction. The sequence was tested in 4 healthy rats and 4 rats with chronic myocardial infarction, approximately 2 months after surgery. As a control, a non-accelerated self-gated multi-slice FLASH sequence with an echo time (TE) of 2.76 ms, 4.5 signal averages, a matrix of 192 × 192, and an acquisition time of 2 min 34 s per slice was used to obtain Cine MRI with 15 frames per heartbeat. Non-accelerated UTE MRI was performed with TE = 0.29 ms, a reconstruction matrix of 192 × 192, and an acquisition time of 3 min 47 s per slice for 3.5 averages. Accelerated imaging with 2×, 4× and 5× undersampled kt-space data was performed with 1 min, 30 and 15 s acquisitions, respectively. UTE Cine images up to 5× undersampled kt-space data could be successfully reconstructed using a compressed sensing algorithm. In contrast to the FLASH Cine images, flow artifacts in the UTE images were nearly absent due to the short echo time, simplifying segmentation of the left ventricular (LV) lumen. LV functional parameters derived from the control and the accelerated Cine movies were statistically identical.

Background: Galectin-3 is a ß-galactoside-binding lectin expressed in most of tissues in normal conditions and overexpressed in myocardium from early stages of heart failure (HF). It is an established biomarker associated with extracellular matrix (ECM) turnover during myocardial remodeling. The aim of this study is to test the ability of (123)I-galectin-3 (IG3) to assess cardiac remodeling in a model of myocardial infarction (MI) using imaging techniques., Methods: Recombinant galectin-3 was labeled with iodine-123 and in vitro binding assays were conducted to test (123)I-galectin-3 ability to bind to ECM targets. For in vivo studies, a rat model of induced-MI was used. Animals were subjected to magnetic resonance and micro-SPETC/micro-CT imaging two (2 W-MI) or four (4 W-MI) weeks after MI. Sham rats were used as controls. Pharmacokinetic, biodistribution, and histological studies were also performed after intravenous administration of IG3., Results: In vitro studies revealed that IG3 shows higher binding affinity (measured as counts per minute, cpm) (p < 0.05) to laminin (2.45 ± 1.67 cpm), fibronectin (4.72 ± 1.95 cpm), and collagen type I (1.88 ± 0.53 cpm) compared to bovine serum albumin (BSA) (0.88 ± 0.31 cpm). Myocardial quantitative IG3 uptake (%ID/g) was higher (p < 0.01) in the infarct of 2 W-MI rats (0.15 ± 0.04%) compared to control (0.05 ± 0.03%). IG3 infarct uptake correlates with the extent of scar (r s = 1, p = 0.017). Total collagen deposition in the infarct (percentage area) was higher (p < 0.0001) at 2 W-MI (24.2 ± 5.1%) and 4 W-MI (30.4 ± 7.5%) compared to control (1.9 ± 1.1%). However, thick collagen content in the infarct (square micrometer stained) was higher at 4 W-MI (20.5 ± 11.2 μm(2)) compared to control (4.7 ± 2.0 μm(2), p < 0.001) and 2 W-MI (10.6 ± 5.1 μm(2), p < 0.05)., Conclusions: This study shows, although preliminary, enough data to consider IG3 as a potential contrast agent for imaging of myocardial interstitial changes in rats after MI. Labeling strategies need to be sought to improve in vivo IG3 imaging, and if proven, galectin-3 might be used as an imaging tool for the assessment and treatment of MI patients.

Purpose: A high-quality, reproducible, multi-slice T2-mapping protocol for the mouse heart is presented., Methods: A T2-prepared sequence with composite 90° and 180° radiofrequency pulses in a segmented MLEV phase cycling scheme was developed. The T2-mapping protocol was optimized using simulations and evaluated with phantoms., Results: Repeatability for determination of myocardial T2 values was assessed in vivo in n = 5 healthy mice on 2 different days. The average baseline T2 of the left ventricular myocardium was 22.5 ± 1.7 ms. The repeatability coefficient for R2 = 1/T2 for measurements at different days was ΔR2 = 6.3 s(−1). Subsequently, T2 mapping was applied in comparison to late-gadolinium-enhancement (LGE) imaging, to assess 1-day-old ischemia/reperfusion (IR) myocardial injury in n = 8 mice. T2 in the infarcts was significantly higher than in remote tissue, whereas remote tissue was not significantly different from baseline. Infarct sizes based on T2 versus LGE showed strong correlation. To assess the time-course of T2 changes in the infarcts, T2 mapping was performed at day 1, 3, and 7 after IR injury in a separate group of mice (n = 16). T2 was highest at day 3, in agreement with the expected time course of edema formation and resolution after myocardial infarction., Conclusion: T2 prepared imaging provides high quality reproducible T2 maps of healthy and diseased mouse myocardium., (2013 Wiley Periodicals, Inc.)

Fluorine MRI ((19) F MRI) is receiving an increasing attention as a viable alternative to proton-based MRI ((1) H MRI) for dedicated application in molecular imaging. The (19) F nucleus has a high gyromagnetic ratio, a 100% natural abundance and is furthermore hardly present in human tissues allowing for hot spot MR imaging. The applicability of (19) F MRI as a molecular and cellular imaging technique has been exploited, ranging from cell tracking to detection and imaging of tumors in preclinical studies. In addition to applications, developing new contrast materials with improved relaxation properties has also been a core research topic in the field, since the inherently low longitudinal relaxation rates of perfluorocarbon compounds result in relatively low imaging efficiency. Borrowed from (1) H MRI, the incorporation of lanthanides, specifically Gd(III) complexes, as signal modulating ingredients in the nanoparticle formulation has emerged as a promising approach to improvement of the fluorine signal. Three different perfluorocarbon emulsions were investigated at five different magnetic field strengths. Perfluoro-15-crown-5-ether was used as the core material and Gd(III)DOTA-DSPE, Gd(III)DOTA-C6-DSPE and Gd(III)DTPA-BSA as the relaxation altering components. While Gd(III)DOTA-DSPE and Gd(III)DOTA-C6-DSPE were favorable constructs for (1) H NMR, Gd(III)DTPA-BSA showed the strongest increase in (19F) R(1). These results show the potential of the use of paramagnetic lipids to increase (19F) R(1) at clinical field strengths (1.5-3 T). At higher field strengths (6.3-14 T), gadolinium does not lead to an increase in (19F) R(1) compared with emulsions without gadolinium, but leads to an significant increase in (19F) R(2). Our data therefore suggest that the most favorable situation for fluorine measurements is at high magnetic fields without the inclusion of gadolinium constructs., (Copyright © 2014 John Wiley & Sons, Ltd.)

In this study, we have performed simulations to address the effects of diffusion encoding parameters, signal-to-noise ratio (SNR) and T2 on skeletal muscle diffusion tensor indices and fiber tracts. Where appropriate, simulations were corroborated and validated by in vivo diffusion tensor imaging (DTI) of human skeletal muscle. Specifically, we have addressed: (i) the accuracy and precision of the diffusion parameters and eigenvectors at different SNR levels; (ii) the effects of the diffusion gradient direction encoding scheme; (iii) the optimal b value for diffusion tensor estimation; (iv) the effects of changes in skeletal muscle T2; and, finally, the influence of SNR on fiber tractography and derived (v) fiber lengths, (vi) pennation angles and (vii) fiber curvatures. We conclude that accurate DTI of skeletal muscle requires an SNR of at least 25, a b value of between 400 and 500 s/mm(2), and data acquired with at least 12 diffusion gradient directions homogeneously distributed on half a sphere. Furthermore, for DTI studies focusing on skeletal muscle injury or pathology, apparent changes in the diffusion parameters need to be interpreted with great care in view of the confounding effects of T2, particularly for moderate to low SNR values., (Copyright © 2013 John Wiley & Sons, Ltd.)

Magnetic resonance imaging (MRI) is a key imaging modality in cancer diagnostics and therapy monitoring. MRI-based tumor detection and characterization is commonly achieved by exploiting the compositional, metabolic, cellular, and vascular differences between malignant and healthy tissue. Contrast agents are frequently applied to enhance this contrast. The last decade has witnessed an increasing interest in novel multifunctional MRI probes. These multifunctional constructs, often of nanoparticle design, allow the incorporation of multiple imaging agents for complementary imaging modalities as well as anti-cancer drugs for therapeutic purposes. The composition, size, and surface properties of such constructs can be tailored as to improve biodistribution and ensure optimal delivery to the tumor microenvironment by passive or targeted mechanisms. Multifunctional MRI probes hold great promise to facilitate more specific tumor diagnosis, patient-specific treatment planning, the monitoring of local drug delivery, and the early evaluation of therapy. This chapter reviews the state-of-the-art and new developments in the application of multifunctional MRI probes in oncology.

The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium-based contrast agents to enhance atherosclerotic plaques in ApoE(-/-) mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer-term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T(1)-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR(664) incorporated in the contrast agents and quantification of tissue and blood Gd-DTPA. Both micelles and liposomes enhanced contrast in T(1)-weighted MR images of plaques in the aortic arch. The average contrast-to-noise ratio increased after liposome or micelle injection to 260 or 280% respectively, at 24 h after injection, compared with a pre-scan. A second wave of maximum contrast enhancement was observed around 60-72 h after injection, which only slowly decreased towards the 1 week end-point. Confocal fluorescence microscopy and whole body fluorescence imaging confirmed MRI-findings of accumulation of micelles and liposomes. Plaque permeation of contrast agents was not strongly dependent on the contrast agent size in this mouse model. Our results show that intraplaque accumulation over time of both contrast agents leads to good plaque visualization for a long period. This inherent intraplaque accumulation might make it difficult to discriminate passive from targeted accumulation. This implies that, in the development of targeted contrast agents on a lipid-based backbone, extensive timing studies are required., (Copyright © 2012 John Wiley & Sons, Ltd.)

Object: Imaging of myocardial infarct composition is essential to assess efficacy of emerging therapeutics. T (2) (*) mapping has the potential to image myocardial hemorrhage and fibrosis by virtue of its short T (2) (*) . We aimed to quantify T (2) (*) in acute and chronic myocardial ischemia/reperfusion (I/R) injury in mice., Materials and Methods: I/R-injury was induced in C57BL/6 mice (n = 9). Sham-operated mice (n = 8) served as controls. MRI was performed at baseline, and 1, 7 and 28 days after surgery. MRI at 9.4 T consisted of Cine, T (2) (*) mapping and late-gadolinium-enhancement (LGE). Mice (n = 6) were histologically assessed for hemorrhage and collagen in the fibrotic scar., Results: Baseline T (2) (*) values were 17.1 ± 2.0 ms. At day 1, LGE displayed a homogeneous infarct enhancement. T (2) (*) in infarct (12.0 ± 1.1 ms) and remote myocardium (13.9 ± 0.8 ms) was lower than at baseline. On days 7 and 28, LGE was heterogeneous. T (2) (*) in the infarct decreased to 7.9 ± 0.7 and 6.4 ± 0.7 ms, whereas T (2) (*) values in the remote myocardium were 14.2 ± 1.1 and 15.6 ± 1.0 ms. Histology revealed deposition of iron and collagen in parallel with decreased T (2) (*) ., Conclusion: T (2) (*) values are dynamic during infarct development and decrease significantly during scar maturation. In the acute phase, T (2) (*) values in infarcted myocardium differ significantly from those in the chronic phase. T (2) (*) mapping was able to confirm the presence of a chronic infarction in cases where LGE was inconclusive. Hence, T (2) (*) may be used to discriminate between acute and chronic infarctions.

Inflammatory activity in atherosclerotic plaque is a risk factor for plaque rupture and atherothrombosis and may direct interventional therapy. Inflammatory activity can be evaluated at the (sub)cellular level using in vivo molecular MRI. This paper reviews recent progress in contrast-enhanced molecular MRI to visualize atherosclerotic plaque inflammation. Various MRI contrast agents, among others ultra-small particles of iron oxide, low-molecular-weight Gd-chelates, micelles, liposomes, and perfluorocarbon emulsions, have been used for in vivo visualization of various inflammation-related targets, such as macrophages, oxidized LDL, endothelial cell expression, plaque neovasculature, MMPs, apoptosis, and activated platelets/thrombus. An enzyme-activatable magnetic resonance contrast agent has been developed to study myeloperoxidase activity in inflamed plaques. Agents creating contrast based on the chemical exchange saturation transfer mechanism were used for thrombus imaging. Transfer of these molecular MRI techniques to the clinic will critically depend on the safety profiles of these newly developed magnetic resonance contrast agents.

Inflammation plays a prominent role in tumor growth. Anti-inflammatory drugs have therefore been proposed as anti-cancer therapeutics. In this study, we determined the anti-angiogenic activity of a single dose of liposomal prednisolone phosphate (PLP-L), by monitoring tumor vascular function and viability over a period of one week. C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma cells. Six animals were PLP-L-treated and six served as control. Tumor tissue and vascular function were probed using MRI before and at three timepoints after treatment. DCE-MRI was used to determine K(trans), v(e), time-to-peak, initial slope and the fraction of non-enhancing pixels, complemented with immunohistochemistry. The apparent diffusion coefficient (ADC), T(2) and tumor size were assessed with MRI as well. PLP-L treatment resulted in smaller tumors and caused a significant drop in K(trans) 48 h post-treatment, which was maintained until one week after drug administration. However, this effect was not sufficient to significantly distinguish treated from non-treated animals. The therapy did not affect tumor tissue viability but did prevent the ADC decrease observed in the control group. No evidence for PLP-L-induced tumor vessel normalization was found on histology. Treatment with PLP-L altered tumor vascular function. This effect did not fully explain the tumor growth inhibition, suggesting a broader spectrum of PLP-L activities.

Molecular imaging based on MRI requires the use of amplification strategies in order to achieve sufficient sensitivity for the detection of low-level molecular markers. Recently, we described a combination of two amplification methods: (i) the use of paramagnetic liposomes that can be prepared with a high payload of Gd(3+)-containing lipid; and (ii) targeting to a cell-surface receptor that can undergo multiple rounds of nanoparticle delivery in the cell, followed by recycling to the cell membrane. Liposome uptake was monitored over a period of 24 h and was found to lead to massive delivery in subcellular compartments. The present study aimed to monitor the longer-term fate of the cell-internalized contrast material by studying its relaxometric properties over 5 days, following an initial 24 h loading period. Circa 25% of the Gd(3+)-content delivered to the cells via integrin-targeted liposomes was lost in the first 24 h, which led to 65 and 77% reductions in R(1) and R(2), respectively, as compared with the original R(1) and R(2) enhancements. This implies that the remaining cell-associated gadolinium had relatively low effective r(1) and r(2) relaxivities. It is proposed that this is due to gradual release of Gd(3+) from the chelate in the cell, followed by sequestration in an MR silent state. Most of the gadolinium internalized by cells following incubation with non-targeted liposomes was released in the 5-day follow-up period., (Copyright © 2010 John Wiley & Sons, Ltd.)

Cardiac MR T(1) mapping is a promising quantitative imaging tool for the diagnosis and evaluation of cardiomyopathy. Here, we present a new preclinical cardiac MRI method enabling three-dimensional T(1) mapping of the mouse heart. The method is based on a variable flip angle analysis of steady-state MR imaging data. A retrospectively triggered three-dimensional FLASH (fast low-angle shot) sequence (3D IntraGate) enables a constant repetition time and maintains steady-state conditions. 3D T(1) mapping of the complete mouse heart could be achieved in 20 min. High-quality, bright-blood T(1) maps were obtained with homogeneous T(1) values (1764 ± 172 ms) throughout the myocardium. The repeatability coefficient of R(1) (1/T(1) ) in a specific region of the mouse heart was between 0.14 and 0.20 s(-1) , depending on the number of flip angles. The feasibility for detecting regional differences in ΔR(1) was shown with pre- and post-contrast T(1) mapping in mice with surgically induced myocardial infarction, for which ΔR(1) values up to 0.83 s(-1) were found in the infarct zone. The sequence was also investigated in black-blood mode, which, interestingly, showed a strong decrease in the apparent mean T(1) of healthy myocardium (905 ± 110 ms). This study shows that 3D T(1) mapping in the mouse heart is feasible and can be used to monitor regional changes in myocardial T(1), particularly in relation to pathology and in contrast-enhanced experiments to estimate local concentrations of (targeted) contrast agent., (Copyright © 2010 John Wiley & Sons, Ltd.)

In vivo molecular imaging with targeted MRI contrast agents will require sensitive methods to quantify local concentrations of contrast agent, enabling not only imaging-based recognition of pathological biomarkers but also detection of changes in expression levels as a consequence of disease development, therapeutic interventions or recurrence of disease. In recent years, targeted paramagnetic perfluorocarbon emulsions have been frequently applied in this context, permitting high-resolution (1)H MRI combined with quantitative (19)F MR imaging or spectroscopy, under the assumption that the fluorine signal is not altered by the local tissue and cellular environment. In this in vitro study we have investigated the (19)F MR-based quantification potential of a paramagnetic perfluorocarbon emulsion conjugated with RGD-peptide to target the cell-internalizing α(ν)β(3)-integrin expressed on endothelial cells, using a combination of (1)H MRI, (19)F MRI and (19)F MRS. The cells took up the targeted emulsion to a greater extent than nontargeted emulsion. The targeted emulsion was internalized into large 1-7 µm diameter vesicles in the perinuclear region, whereas nontargeted emulsion ended up in 1-4 µm diameter vesicles, which were more evenly distributed in the cytoplasm. Association of the targeted emulsion with the cells resulted in different proton longitudinal relaxivity values, r(1), for targeted and control nanoparticles, prohibiting unambiguous quantification of local contrast agent concentration. Upon cellular association, the fluorine R(1) was constant with concentration, while the fluorine R(2) increased nonlinearly with concentration. Even though the fluorine relaxation rate was not constant, the (19)F MRI and (19)F MRS signals for both targeted nanoparticles and controls were linear and quantifiable as function of nanoparticle concentration., (Copyright © 2010 John Wiley & Sons, Ltd.)

Treatment of disease can only be effective when timely and accurate diagnosis of the pathology is achieved. More precise diagnosis can be accomplished if the underlying molecular processes involved in the pathology can be imaged in vivo. This is the field of molecular imaging, which aims to visualize cellular function and molecular processes in living organisms in a non-invasive way. With that aim, molecular markers are specifically targeted by imaging contrast agents. Molecular MRI needs powerful targeted contrast agents. For that purpose, target-specific gadolinium-containing paramagnetic and superparamagnetic, iron oxide-based micelles have been developed. Micelles are lipid-based nanoparticles which are biocompatible and carry a high payload of MR contrast-generating agent. The coupling of high-affinity ligands makes the micelles target-specific. Additionally, this lipid-based micelle platform allows for incorporation of contrast generating molecules for other imaging modalities, e.g., fluorescence or nuclear imaging. This permits applications for multiple imaging modalities, making micelles a highly versatile contrast agent.

Quantum dots exhibit unique optical properties for bioimaging purposes. We have previously developed quantum dots with a paramagnetic and functionalized coating and have shown their potential for molecular imaging purposes. In the current mini-review we summarize the synthesis procedure, the in vitro testing and, importantly, the in vivo application for multimodal molecular imaging of tumor angiogenesis.

Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.

Objectives: We report here on the pre-clinical MRI characterization of an apoE-/- mouse model of stable and vulnerable carotid artery atherosclerotic plaques, which were induced by a tapered restriction (cast) around the artery. Specific focus was on the quantification of the wall shear stress, which is considered a key player in the development of the plaque phenotype., Materials and Methods: In vivo MRI was performed at 9.4 T. The protocol consisted of time-of-flight angiography, high-resolution T1- and T2-weighted black-blood imaging and phase-contrast flow velocity imaging as function of time in the cardiac cycle. Wall shear stress was determined by fitting the flow profile to a quadratic polynomial., Results: Time-of-flight angiography confirmed preservation of blood flow through the carotid arteries in all cases. T1- and T2-weighted MRI resulted in high-resolution images in which the position of the cast, luminal narrowing introduced by cast and plaque, as well as the arterial wall could be well identified. Laminar flow with low wall shear stress (11.2+/- 5.2 Pa) was measured upstream to the cast at the position of the vulnerable plaque. Downstream to the cast at the position of the stable plaque, the apparent velocities were low, which is consistent with vortices and an oscillatory nature of the flow., Conclusions: Flow velocities and wall shear stress were successfully measured in this mouse model of stable and unstable plaque. The presented tools can be used to provide valuable insights in the pathogenesis of atherosclerosis.

Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.

Apoptosis, or programmed cell death, is a morphologically and biochemically distinct form of cell death, which together with proliferation plays an important role in tissue development and homeostasis. Insufficient apoptosis is important in the pathology of various disorders such as cancer and autoimmune diseases, whereas a high apoptotic activity is associated with myocardial infarction, neurodegenerative diseases, and advanced atherosclerotic lesions. Consequently, apoptosis is recognized as an important therapeutic target, which should be either suppressed, e.g., during an ischemic cardiac infarction, or promoted, e.g., in the treatment of cancerous lesions. Imaging tools to address location, amount, and time course of apoptotic activity non-invasively in vivo are therefore of great clinical use in the evaluation of such therapies. This chapter reviews current literature and new developments in the application of nanoparticles for non-invasive apoptosis imaging. Focus is on functionalized nanoparticle contrast agents for MR imaging and bimodal nanoparticle agents that combine magnetic and fluorescent properties.

Modern medicine has greatly benefited from recent dramatic improvements in imaging techniques. The observation of physiological events through interactions manipulated at the molecular level offers unique insight into the function (and dysfunction) of the living organism. The tremendous advances in the development of nanoparticulate molecular imaging agents over the past decade have made it possible to noninvasively image the specificity, pharmacokinetic profiles, biodistribution, and therapeutic efficacy of many novel compounds. Several types of nanoparticles have demonstrated utility for biomedical purposes, including inorganic nanocrystals, such as iron oxide, gold, and quantum dots. Moreover, natural nanoparticles, such as viruses, lipoproteins, or apoferritin, as well as hybrid nanostructures composed of inorganic and natural nanoparticles, have been applied broadly. However, among the most investigated nanoparticle platforms for biomedical purposes are lipidic aggregates, such as liposomal nanoparticles, micelles, and microemulsions. Their relative ease of preparation and functionalization, as well as the ready synthetic ability to combine multiple amphiphilic moieties, are the most important reasons for their popularity. Lipid-based nanoparticle platforms allow the inclusion of a variety of imaging agents, ranging from fluorescent molecules to chelated metals and nanocrystals. In recent years, we have created a variety of multifunctional lipid-based nanoparticles for molecular imaging; many are capable of being used with more than one imaging technique (that is, with multimodal imaging ability). These nanoparticles differ in size, morphology, and specificity for biological markers. In this Account, we discuss the development and characterization of five different particles: liposomes, micelles, nanocrystal micelles, lipid-coated silica, and nanocrystal high-density lipoprotein (HDL). We also demonstrate their application for multimodal molecular imaging, with the main focus on magnetic resonance imaging (MRI), optical techniques, and transmission electron microscopy (TEM). The functionalization of the nanoparticles and the modulation of their pharmacokinetics are discussed. Their application for molecular imaging of key processes in cancer and cardiovascular disease are shown. Finally, we discuss a recent development in which the endogenous nanoparticle HDL was modified to carry different diagnostically active nanocrystal cores to enable multimodal imaging of macrophages in experimental atherosclerosis. The multimodal characteristics of the different contrast agent platforms have proven to be extremely valuable for validation purposes and for understanding mechanisms of particle-target interaction at different levels, ranging from the entire organism down to cellular organelles.

In recent years, numerous Gd(3+)-based contrast agents have been developed to enable target-specific MR imaging of in vivo processes at the molecular level. The combination of powerful contrast agents and amplification strategies, aimed at increasing the contrast agent dose at the target site, is an often-used strategy to improve the sensitivity of biomarker detection. One such amplification mechanism is to target a disease-specific cell membrane receptor that can undergo multiple rounds of internalization following ligand binding and thus shuttle a sizeable amount of contrast agent into the target cell. An example of such a membrane receptor is the alpha(nu)beta(3) integrin. The goal of this study was to investigate the consequences of this amplification approach for the T(1)- and T(2)-shortening efficacy of a paramagnetic contrast agent. Cultured endothelial cells were incubated with paramagnetic liposomes that were conjugated with a cyclic RGD-peptide to enable internalization by means of the alpha(nu)beta(3) integrin receptor. Non-targeted liposomes served as a control. This study showed that alpha(nu)beta(3) targeting dramatically increased the uptake of paramagnetic liposomes. This targeting strategy, however, strongly influenced both the longitudinal and transverse relaxivity of the internalized paramagnetic liposomes., ((c) 2009 Wiley-Liss, Inc.)

The goal of this work was to elaborate a model describing the effective longitudinal relaxation rate constant R(1) for (1)H(2)O in three cellular compartments experiencing possible equilibrium water exchange, and to apply this model to explain the effective R(1) dependence on the overall concentration of a cell-internalized Gd(3+)-based contrast agent (CA). The model voxel comprises three compartments representing extracellular, cytoplasmic, and vesicular (e.g., endosomal, lysosomal) subcellular spaces. Relaxation parameters were simulated using a modified Bloch-McConnell equation including magnetization exchange between the three compartments. With the model, several possible scenarios for internalized CA distribution were evaluated. Relaxation parameters were calculated for contrast agent restricted to the cytoplasmic or vesicular compartments. The size or the number of CA-loaded vesicles was varied. The simulated data were then separately fitted with empirical mono- and biexponential inversion recovery expressions. The voxel CA-concentration dependencies of R(1) can be used to qualitatively and quantitatively understand a number of different experimental observations reported in the literature. Most important, the simulations reproduced the relaxivity "quenching" for cell-internalized contrast agent that has been observed., ((c) 2009 Wiley-Liss, Inc.)

Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel alphavbeta3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.