Osmotically shrunken LIPOCEST agents: an innovative class of magnetic resonance imaging contrast media based on chemical exchange saturation transfer

The peculiar properties of osmotically shrunken liposomes acting as magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) contrast agents have been investigated. Attention has been primarily devoted to assessing the contribution arising from encapsulated and incorporated paramagnetic lanthanide(III)-based shift reagents in determining the chemical shift of the intraliposomal water protons, which is a relevant factor for generating the CEST contrast. It is demonstrated that a highly shifted resonance for the encapsulated water can be attained by increasing the percentage of the amphiphilic shift reagent incorporated in the liposome bilayer. It is also demonstrated that the shift contribution arising from the bulk magnetic susceptibility can be optimized through the modulation of the osmotic shrinkage. In terms of sensitivity, it is shown that the saturation transfer efficiency can be significantly improved by increasing the size of the vesicle, thus allowing a high number of exchangeable protons to be saturated. In addition, the role played by the intensity of the saturating radiofrequency field has also been highlighted

Influence of cell-internalization on relaxometric, optical and compositional properties of targeted paramagnetic quantum dot micelles

Quantum dot micelles (pQDs) with a paramagnetic coating are promising nanoparticles for bimodal molecular imaging. Their bright fluorescence allows for optical detection, while their Gd payload enables visualization with contrast-enhanced MRI. A popular approach in molecular MRI is the targeting of abundantly expressed cell surface receptors. Ligand-receptor binding often results in cell internalization of the targeted contrast agent. The interpretation of molecular imaging with pQDs therefore requires knowledge about the consequences of cellular internalization for their relaxometric, optical and compositional properties. To study these, Cd-containing core-shell-shell QDs coated with a monolayer of lipids, of which 50¿mol% was a Gd-containing lipid, were incubated with human umbilical vein-derived endothelial cells (HUVECs) for up to 24¿h. a¿ß3-integrin targeted (RGD) and non-targeted (NT) pQDs were compared. pQDs uptake was monitored by fluorescence microscopy, FACS, ICP-MS, relaxometry and MRI. Cell-associated pQDs displayed longitudinal relaxation rates and fluorescent intensities which were linear with the cell-associated Gd and Cd concentrations, implying that the Gd and Cd uptake by HUVECs can be quantified using relaxometric and optical measurements, respectively. However, the Gd-to-Cd molar ratio in pellets of pQD-incubated cells was consistently higher than the Gd-to-Cd molar ratio of the pQDs as prepared. It is proposed that this increase in Gd-to-Cd molar ratio was due to non-specific lipid-transfer between the pQDs and the cellular membranes. These findings show that, in the case of contrast agents that are formed by non-covalent interactions, experimental procedures are needed with which representative components of the probes can be monitore

Molecular MR imaging of collagen in mouse atherosclerosis by using paramagnetic CNA35 micelles

Magnetic resonance imaging (MRI) is increasingly used in biomedicine to visualize plaques in the walls of major arteries in relation to atherosclerosis, the prime cause of myocardial infarction and ischemic stroke. The present study aims to explore the utility of contrast-enhanced MRI for improving the specificity of the MRI evaluation of atherosclerotic plaques with the use of a Gd-based paramagnetic contrast agent that is targeted to collagen. Collagen is a major component of the extracellular matrix and as such plays an important role in the stability of atherosclerotic plaques. Micelles were made with lipid containing 45 mol-% Gd for MRI detection and a low mol fraction of fluorescent lipid for fluorescence microscopic analysis. Collagen-targeted, functional micelles were prepared by conjugation of the CNA35 protein, while nonfunctional control micelles were conjugated with a mutated version of the protein. The micelles were characterized with respect to their magnetic, biochemical, and biophysical properties. Atherosclerotic plaques were induced in the right carotid artery of apo-E knock-out mice by surgical placement of a tapered polymeric cast. In vivo MRI was performed at 6.3 Tesla before and up to 24 h after intravenous injection of paramagnetic micelles (50 µmol Gd kg -1). MRI revealed the strongest signal enhancements by CNA35 micelles. At early time points after injection of CNA35 micelles, contrast enhancement was higher in the collagen-richer lesions compared to that in the collagen-poorer lesions. Confocal laser scanning microscopy confirmed co-localization of CNA35 micelles and collagen in the plaques. We have demonstrated molecular MR imaging of collagen in experimental atherosclerosis by using a CNA35-functionalized micellar contrast agent

Morphology, binding behavior and MR-properties of paramagnetic collagen-binding liposomes

Collagen is an important component of the extracellular matrix (ECM) and plays an important role in normal tissue maturation and in pathological processes such as atherosclerosis and myocardial infarction. The diagnostics of the latter diseases using MRI could strongly benefit from the use of collagen-specific contrast agents. The current study aimed to develop a bimodal liposomal MR contrast agent that was functionalized with CNA35, a collagen adhesion protein of the Staphylococcus aureus bacterium. The liposomes were characterized in terms of CNA35 protein conjugation and loading. The overall morphology was assessed with DLS and cryo-TEM, while cryo-TEM tomography was used to visualize the protein coverage of the liposomes. The binding properties of the contrast agent were investigated using a fluorescence assay based on the rhodamine content of the liposomes. The bulk relaxivity was determined using regular relaxometry while the MR-properties of liposomes in their bound state were studied using NMR depth profiling. This CNA35 functionalized contrast agent and the set of in vitro experiments we performed indicate the potential of this technology for in vivo molecular imaging of collagen

Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells

Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo. Copyright 2009 John Wiley & Sons, Ltd

Diffusion tensor imaging of left ventricular remodeling in response to myocardial infarction in the mouse

The cardiac muscle architecture lies at the basis of the mechanical and electrical properties of the heart, and dynamic alterations in fiber structure are known to be of prime importance in healing and remodeling after myocardial infarction. In this study, left ventricular remodeling was characterized using diffusion tensor imaging (DTI) in a mouse model of myocardial infarction. Myocardial infarction was induced in mice by permanent ligation of the left anterior descending coronary artery. Serial ex vivo DTI measurements were performed 7, 14, 28, and 60 days after ligation. Apparent diffusion coefficient, fractional anisotropy, the three eigenvalues of the diffusion tensor, and the myofiber disarray served as readout parameters. After myocardial infarction, the mouse hearts displayed extreme wall thinning in the infarcted area, which covered large parts of the apex and extended into the free wall up to the equator. Average heart mass increased by 70% 7-60 days after infarction. Histological analysis showed that the infarct at 7 days consisted of unstructured tissue with residual necrosis and infiltration of macrophages and myofibroblasts. At 14 days after infarction, the necrotic tissue had disappeared and collagen fibers were starting to appear. From 28 to 60 days, the infarct had fully developed into a mature scar. DTI parameters showed dynamic changes as a function of time after infarction. The apparent diffusion coefficient in the infarcted region was lower than in remote regions and increased as a function of time after infarction. The fractional anisotropy was higher in the infarcted region and was maximum at 28 days, which was attributed to the development of structured collagen fibers. Myofiber disarray, which was analyzed by considering the alignment of fibers in neighboring voxels, was significantly higher in infarcted regions. DTI provides a valuable non-destructive tool for characterizing structural remodeling in diseased myocardium