Contrast enhancement by differently sized paramagnetic MRI contrast agents in mice with two phenotypes of atherosclerotic plaque

Interest in the use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of three differently sized gadolinium-based contrast agents to permeate different mouse plaque phenotypes was evaluated with MRI. A tapered cast was implanted around the right carotid artery of apoE-/- mice to induce two different plaque phenotypes: a thin cap fibroatheroma (TCFA) and a non-TCFA lesion. Both plaques were allowed to develop over 6 and 9 weeks, leading to an intermediate and advanced lesion, respectively. Signal enhancement in the carotid artery wall, following intravenous injection of Gd-HP-DO3A as well as paramagnetic micelles and liposomes was evaluated. In vivo T1-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy and correlated to lesion phenotype. The two smallest contrast agents, i.e. Gd-HP-DO3A and micelles, were found to enhance contrast in T1-weighted MR images of all investigated plaque phenotypes. Maximum contrast enhancement ranged between 53 and 70% at 6¿min after injection of Gd-HP-DO3A with highest enhancement and longest retention in the non-TCFA lesion. Twenty-four hours after injection of micelles maximum contrast enhancement ranged between 24 and 35% in all plaque phenotypes. Administration of the larger liposomes did not cause significant contrast enhancement in the atherosclerotic plaques. Confocal fluorescence microscopy confirmed the MRI-based differences in plaque permeation between micelles and liposomes. Plaque permeation of contrast agents was strongly dependent on size. Our results implicate that, when equipped with targeting ligands, liposomes are most suitable for the imaging of plaque-associated endothelial markers due to low background enhancement, whereas micelles, which accumulate extravascularly on a long timescale, are suited for imaging of less abundant markers inside plaques. Low molecular weight compounds may be employed for target-specific imaging of highly abundant extravascular plaque-associated target

Molecular and functional MRI of cardiac remodeling after myocardial infarction

Myocardial infarction is the leading cause of death world-wide. It is characterized by cardiomyocyte cell death resulting from local oxygen deprivation caused by obstructions in the coronary microcirculation. The heart has very limited potential to regenerate new myocardium, and instead the dead cardiomyocytes are replaced by a non-contractile fibrotic scar tissue. This is accompanied by a progressive cardiac remodeling process, resulting in left ventricular dilation and wall thinning of the infarcted myocardium and gradual infarct expansion into non-ischemic myocardium. Many patients, who survive the initial acute stage of cardiac infarction, however, ultimately develop heart failure because of the inadequate long-term response of the heart to the ischemic insult. Therefore, there is an urgent need for novel treatment strategies that can substantially improve the long-term prognosis after myocardial infarction. For successful clinical implementation of new therapies non-invasive readouts are crucial to 1) select patients for whom therapy is expected to be effective and 2) monitor a patient’s response to therapy. In this thesis, novel therapies and readouts were developed and their preclinical evaluation was performed in a mouse model of myocardial infarction. Magnetic resonance imaging (MRI) is an attractive non-invasive imaging technique that can be used to evaluate global and regional cardiac morphology and contractile function, but can also provide a detailed characterization of the myocardium at the cellular and even molecular level by using MRI contrast agents. Contrast agents can generate contrast between infarcted and remote myocardium either by differences in the local passive distribution pattern or by actively targeting them towards specific cells, proteins or enzymes involved in myocardial infarction. An attractive contrast agent platform are nanometer-sized particles. To stimulate reparative processes in the infarct, efficient delivery and retention of therapeutic agents is desired. This might be achieved by encapsulation of drugs in nanoparticles. To non-invasively evaluate the efficiency of nanoparticle trafficking to the infarct, paramagnetic contrast agents can be incorporated in these nanoparticles for in vivo visualization by MRI. Two types of paramagnetic and fluorescent lipid-based nanoparticles, namely micelles and liposomes, were applied in mice with acute and chronic myocardial infarction to determine their cardiac distribution pattern, and thus their possible utility as drug delivery vehicle to infarcted myocardium. In both acute and chronic infarcts, micelles permeated the entire necrotic myocardium, whereas liposomes displayed slower and more restricted extravasation from the vasculature. Therefore, paramagnetic micelles and liposomes are attractive nanocarriers for transporting distinct types of drugs to the infarct. Importantly, the successful in vivo MRI monitoring of the delivery and spatial distribution of paramagnetic nanocarriers is a promising tool for optimizing drug delivery to infarcted myocardium in preclinical pharmacological research. Moreover, paramagnetic lipid-based nanoparticles are also interesting constructs for molecular MR imaging of processes ongoing in the infarct at the cellular or molecular level to improve patient diagnosis and monitoring. For this purpose, nanoparticles must be functionalized with ligands that can bind to a specific cell receptor, protein or enzyme. We conjugated ICAM-1 specific antibodies to paramagnetic liposomes to explore their ability to visualize ICAM-1 upregulation on the endothelium of the vasculature in the infarcted myocardium and its border zones using in vivo MRI. ICAM-1 expression on blood vessels is critical for the recruitment of leukocytes, which can inflict additional damage to the myocardium. First, the in vitro binding behavior of ICAM-1 targeted liposomes to ICAM-1 expressing endothelial cells was evaluated. ICAM-1 targeted liposomes could differentiate between low and high levels of endothelial ICAM-1 expression with MRI. In addition, ICAM-1 binding was observed in the competing presence of leukocytes and when shear stress was applied to mimic blood flow. These promising in vitro results encouraged follow up in vivo studies. In healthy mice, the circulation half-life of ICAM-1 targeted liposomes was short compared to the half-life of non-specific liposomes, indicating binding to constitutively expressed ICAM-1. Indeed, massive binding of ICAM-1 targeted liposomes to ICAM-1 expressing lung endothelium was observed. In mice with myocardial infarction, ICAM-1 binding liposomes were mainly associated with the vasculature in the infarct periphery and borders, which are sites with highly increased ICAM-1 expression. This was deduced from ex vivo fluorescence microscopy. However, this targeting effect did not create specific in vivo signal enhancement in MR images of the infarcted heart, indicating that the sensitivity of in vivo MRI to detect ICAM-1 upregulation with this contrast agent formulation was not sufficient. A promising approach to improve the healing of the infarct is cell transplantation, which might generate new myocardium. This thesis describes a comparison of three distinct types of cell treatments – namely intra-infarct injection of rhythmically contractile cardiac progenitor cells, contractile myoblasts or non-contractile mesenchymal stem cells – to gain more insight in the requirements of the ideal cell type for myocardial regeneration. Conventional late gadolinium enhancement and cinematographic MRI techniques were used to monitor the effects of cell transplantation on infarct size and cardiac function. Cardiac progenitor cells were found to substantially improve the condition of the heart and were the only cell type leading to decreased infarct size. In addition, they reduced wall thinning of the infarct center and border zones and importantly they improved the contractile function in the infarct borders. Therefore, the ability of transplanted cells to adopt a contractile cardiomyocyte phenotype seems crucial to improve the local contractile function. Nevertheless, the survival of injected cells in the infarct center must be improved to further enhance the infarct contractility. To summarize, this thesis describes studies in mice on the development of novel diagnostic and therapeutic approaches for the treatment of myocardial infarction based on non-invasive MRI techniques. This might ultimately improve the monitoring and treatment of patients to decrease the mortality after myocardial infarction

Towards efficient cancer immunotherapy: advances in developing artificial antigen-presenting cells

Contains fulltext : 137111.pdf (publisher's version ) (Open Access)Active anti-cancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells (APCs). Therapy with autologous natural APCs is costly and time-consuming and results in variable outcomes in clinical trials. Therefore, development of artificial APCs (aAPCs) has attracted significant interest as an alternative. We discuss the characteristics of various types of acellular aAPCs, and their clinical potential in cancer immunotherapy. The size, shape, and ligand mobility of aAPCs and their presentation of different immunological signals can all have significant effects on cytotoxic T cell activation. Novel optimized aAPCs, combining carefully tuned properties, may lead to efficient immunomodulation and improved clinical responses in cancer immunotherapy

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

Contains fulltext : 108401.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells

ACCIRT/WRC Newsletter [30 April, 2014]

Cancer immunotherapy critically relies on the efficient presentation of tumor antigens to T-cells to elicit a potent anti-tumor immune response aimed at life-long protection against cancer recurrence. Recent advances in the nanovaccine field have now resulted in formulations that trigger strong anti-tumor responses. Nanovaccines are assemblies that are able to present tumor antigens and appropriate immune-stimulatory signals either directly to T-cells or indirectly via antigen-presenting dendritic cells. This review focuses on important aspects of nanovaccine design for dendritic cells, including the synergistic and cytosolic delivery of immunogenic compounds, as well as their passive and active targeting to dendritic cells. In addition, nanoparticles for direct T-cell activation are discussed, addressing features necessary to effectively mimic dendritic cell/T-cell interactions

Mouse myocardial first-pass perfusion MR imaging

A first-pass myocardial perfusion sequence for mouse cardiac MRI is presented. A segmented ECG-triggered acquisition combined with parallel imaging acceleration was used to capture the first pass of a Gd-DTPA bolus through the mouse heart with a temporal resolution of 300–400 msec. The method was applied in healthy mice (N = 5) and in mice with permanent occlusion of the left coronary artery (N = 6). Baseline semiquantitative perfusion values of healthy myocardium showed excellent reproducibility. Infarct regions revealed a significant decrease in the semiquantitative myocardial perfusion values (0.05 ± 0.02) compared to remote myocardium (0.20 ± 0.04). Myocardial areas of decreased perfusion correlated well to infarct areas identified on the delayed-enhancement scans. This protocol is a valuable addition to the mouse cardiac MRI toolbox for preclinical studies of ischemic heart disease

Three-dimensional T1 mapping of the mouse heart using variable flip anglesteady-state MR imaging

Cardiac MR T1 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 T1 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 T1 mapping of the complete mouse heart could be achieved in 20¿min. High-quality, bright-blood T1 maps were obtained with homogeneous T1 values (1764¿±¿172¿ms) throughout the myocardium. The repeatability coefficient of R1 (1/T1) 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 ¿R1 was shown with pre- and post-contrast T1 mapping in mice with surgically induced myocardial infarction, for which ¿R1 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 T1 of healthy myocardium (905¿±¿110¿ms). This study shows that 3D T1 mapping in the mouse heart is feasible and can be used to monitor regional changes in myocardial T1, particularly in relation to pathology and in contrast-enhanced experiments to estimate local concentrations of (targeted) contrast agent

Distribution of lipid-based nanoparticles to infarcted myocardium with potential application for MRI-monitored drug delivery

Item does not contain fulltextAdverse cardiac remodeling after myocardial infarction ultimately causes heart failure. To stimulate reparative processes in the infarct, efficient delivery and retention of therapeutic agents is desired. This might be achieved by encapsulation of drugs in nanoparticles. The goal of this study was to characterize the distribution pattern of differently sized long-circulating lipid-based nanoparticles, namely micelles (~15 nm) and liposomes (~100 nm), in a mouse model of myocardial infarction (MI). MI was induced in mice (n=38) by permanent occlusion of the left coronary artery. Nanoparticle accumulation following intravenous administration was examined one day and one week after surgery, representing the acute and chronic phase of MI, respectively. In vivo magnetic resonance imaging of paramagnetic lipids in the micelles and liposomes was employed to monitor the trafficking of nanoparticles to the infarcted myocardium. Ex vivo high-resolution fluorescence microscopy of fluorescent lipids was used to determine the exact location of the nanoparticles in the myocardium. In both acute and chronic MI, micelles permeated the entire infarct area, which renders them very suited for the local delivery of cardioprotective or anti-remodeling drugs. Liposomes displayed slower and more restricted extravasation from the vasculature and are therefore an attractive vehicle for the delivery of pro-angiogenic drugs. Importantly, the ability to non-invasively visualize both micelles and liposomes with MRI creates a versatile approach for the development of effective cardioprotective therapeutic interventions

Contrast enhancement by differently sized paramagnetic MRI contrast agents in mice with two phenotypes of atherosclerotic plaque

Interest in the use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of three differently sized gadolinium-based contrast agents to permeate different mouse plaque phenotypes was evaluated with MRI. A tapered cast was implanted around the right carotid artery of apoE-/- mice to induce two different plaque phenotypes: a thin cap fibroatheroma (TCFA) and a non-TCFA lesion. Both plaques were allowed to develop over 6 and 9 weeks, leading to an intermediate and advanced lesion, respectively. Signal enhancement in the carotid artery wall, following intravenous injection of Gd-HP-DO3A as well as paramagnetic micelles and liposomes was evaluated. In vivo T1-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy and correlated to lesion phenotype. The two smallest contrast agents, i.e. Gd-HP-DO3A and micelles, were found to enhance contrast in T1-weighted MR images of all investigated plaque phenotypes. Maximum contrast enhancement ranged between 53 and 70% at 6¿min after injection of Gd-HP-DO3A with highest enhancement and longest retention in the non-TCFA lesion. Twenty-four hours after injection of micelles maximum contrast enhancement ranged between 24 and 35% in all plaque phenotypes. Administration of the larger liposomes did not cause significant contrast enhancement in the atherosclerotic plaques. Confocal fluorescence microscopy confirmed the MRI-based differences in plaque permeation between micelles and liposomes. Plaque permeation of contrast agents was strongly dependent on size. Our results implicate that, when equipped with targeting ligands, liposomes are most suitable for the imaging of plaque-associated endothelial markers due to low background enhancement, whereas micelles, which accumulate extravascularly on a long timescale, are suited for imaging of less abundant markers inside plaques. Low molecular weight compounds may be employed for target-specific imaging of highly abundant extravascular plaque-associated target