Jeff W. M. Bulte

Synthesis and relaxometry of high-generation (G = 5, 7, 9, and 10) PAMAM dendrimer-DOTA-gadolinium chelates †

A series of high‐generation (G) ethylenediamine‐core polyamidoamine (PAMAM) dendrimers corresponding to G = 5, 7, 9, and 10 were conjugated with the bifunctional chelate 2‐(4‐isothiocyanatobenzyl)‐1,4,7,10‐tetraazacyclododecane‐N,N′,N”,N”‐tetraacetate (p‐SCN‐Bz‐DOTA). Gadolinium (III) ion was added to the macromolecules, and the 1/T1 and 1/T2 NMRD profiles were measured at 3°, 23°, and 37°C. The synthesis resulted in preparations that ranged from an average of 127 chelates and 96 Gd3+ ions per G = 5 dendrimer to an average of 3727 chelates and1860 Gd3+ ions per G = 10 dendrimer. At 20 MHz and 23°C, the 1/T1 ion relaxivity increased from 30 mM‐1s ‐1 for the G = 5 to 35 mM‐1 s‐1 for the G = 7 PAMAM dendrimer‐DOTA‐Gd, reaching a plateau at 36 mM‐1 s‐1 for the G = 9 and G = 10 dendrimers. A similar plateau was observed for 1/T2 with values of 36 mM‐1 s‐1 for G = 5, 42 mM‐1 s‐1 for G = 7, and 45 mM‐1 s‐1 for the G = 9 and G = 10 dendrimers. This “saturation” of ion relaxivity for high‐generation dendrimers occurred over the entire frequency range studied. The 1/T1 and 1/T2 relaxivities decreased as the temperature decreased for each generation of dendrimer studied, implying that slow water exchange of bound water molecules with the bulk solvent limits the relaxivity. In such circumstances, increases in the rotational correlation time of the macromolecules associated with higher generations of dendrimer does not result in significant increases in the ion relaxivity. Although the ion relaxivity does not increase, the total molecular relaxivities increased from 2880 mM‐1 s‐1 to 66960 mM‐1 s‐1 for the G = 5 to the G = 10 dendrimer. The current findings are relevant for the design of high‐generation dendrimer‐based receptor agents. J. Magn. Reson. Imaging 1999; 9:348–352.

Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents.

Mammalian stem cells or other cells are being considered for use for infusion or transplantation into tissue for purposes of repair or for revascularization or therapeutic approaches (i.e., genetically altered cells) (1–5). Dextrancoated superparamagnetic iron oxide (SPIO) nanoparticles, a distinct class of MR contrast agents, cannot be used to efficiently label stem cells or other mammalian cells in vitro in their native unmodified form (6–8). Previously, we demonstrated that by conjugating antigenspecific internalizing monoclonal antibodies to the surface, dextran coating cells could be magnetically labeled during their normal expansion in culture (6). This magnetic labeling approach is limited because it requires the availability of an internalizing monoclonal antibody that recognizes a specific cell surface antigen. It is suitable only for labeling of cells that express the targeted receptor and is commonly species specific. Other approaches have involved the synthesis and modification of ultrasmall SPIO (USPIO or MION) particles with tat-proteins facilitating the incorporation into the cells, although this involves also a (synthetic) protein derivative (7,8). An alternative approach is the complex synthesis of a superparamagnetic iron oxide (SPIO) coated with dendrimers or “magnetodendrimers”, which will non-specifically label most mammalian cells (9). The dendrimer coating of the iron oxide nanoparticles provides the needed high affinity for cellular membranes for the construction of suitable cellular contrast agents, as dendrimers are commonly used as non-viral transfection agents (10,11). Magnetodendrimers can be used to efficiently and non-specifically label mammalian stem cells and cancer cells (12,13), however, they are not widely available or approved by the Food and Drug Administration (FDA). Over the past 10 years there has been significant research in developing new transfection agents (TA) including cationic peptides, dendrimers, poly-amines and lipids for nonviral transfection of DNA into the nucleus (10,14,15). These transfection agents are being developed to overcome the problem of the endosomal capture of the TA-DNA complex and inefficient release of the targeted material into the nucleus (14,15). TA’s are macromolecules with molecular weights from 1 to 10 kilo Dalton possessing an electrostatic charge. Based upon efficient labeling of mammalian cells by magnetodendrimers (12,13), we hypothesize that commercially available macromolecular transfection agents would coat via electrostatic interaction with dextran-coated iron oxide MR contrast agents and chaperon these nanoparticles into cells. We present here the magnetic labeling results of combining dendrimers and other commercially available TA’s with (FDA-approved) dextran-coated iron oxide MR contrast agents (Feridex and MION-46L). Acad Radiol 2002; 9(suppl 2):S484–S487

Improved molecular imaging contrast agent for detection of human thrombus

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus‐specific agent. Effective molecular imaging has been previously demonstrated with fibrin‐targeted Gd‐DTPA‐bis‐oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin‐targeted paramagnetic formulation, Gd‐DTPA‐phosphatidylethanolamine (PE), was compared with Gd‐DTPA‐BOA at 0.05‐4.7 T. Ion‐ and particle‐based r1 relaxivities (1.5 T) for Gd‐DTPA‐PE (33.7 (s*mM)‐1 and 2.48 × 106 (s*mM)‐1, respectively) were about twofold higher than for Gd‐DTPA‐BOA, perhaps due to faster water exchange with surface gadolinium. Gd‐DTPA‐PE nanoparticles bound to thrombus surfaces via anti‐fibrin antibodies (1H10) induced 72% ± 5% higher change in R1 values at 1.5 T (ΔR1 = 0.77 ± 0.02 1/s) relative to Gd‐DTPA‐BOA (ΔR1 = 0.45 ± 0.02 1/s). These studies demonstrate marked improvement in a fibrin‐specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack. Magn Reson Med 50:411–416, 2003.

MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain

Stem cell transplantation is being explored as a new paradigm for the treatment of demyelinating diseases. Magnetically labeled multipotential neural precursor cells were transplanted into the ventricles of rats with acute experimental allergic encephalomyelitis (EAE) and high‐resolution (microscopic) MR images were obtained ex vivo. Migration patterns of live cells into periventricular white matter structures could be easily visualized, with a good correlation of the corresponding histopathology. The present results confirm that MR cell tracking can be used to guide the development of successful transplantation protocols. Magn Reson Med 50:201–205, 2003.

Preparation, relaxometry, and biokinetics of PEGylated magnetoliposomes as MR contrast agent

Abstract We prepared PEGylated magnetoliposomes (ML-PEG) with a diameter of 40xa0nm and containing 1–6 superparamagnetic iron oxide crystals per vesicle. The T 1 and T 2 relaxivities were 3 and 240 s −1 /mM at 1.5xa0T and 37°C. In rats, ML-PEG showed a biexponential clearance with a long blood half-life of 53.2±13.2xa0min. Using MR imaging, ML-PEG was found to have excellent properties as a bone marrow-seeking MR contrast agent.

Short‐ vs. long‐circulating magnetoliposomes as bone marrow‐seeking MR contrast agents

We evaluated the relaxation enhancement and biodistribution of short‐ vs. long‐circulating magnetoliposomes as a new contrast agent for magnetic resonance (MR) imaging of bone marrow. Magnetoliposomes with (ML‐PEG) and without (ML) incorporation of polyethylene glycol (PEG, Mw 2000) were prepared, measuring 40 nm in diameter with 1–6 iron oxide crystals/vesicle. PEGylation selectively enhanced the T2 relaxivity of magnetoliposomes by 10% to 15%, with R1 and R2 values of 3 and 240 s‐1/mM at 1.5 T and 37°C. ML (n = 6) and ML‐PEG (n = 6) preparations were administered IV into young (6–8 weeks old) and adult (>1 year old) Sprague‐Dawley rats at 100 μmol Fe/kg. PEGylation increased blood half‐life (P < 0.05 for t > 30 minutes), following a biexponential clearance with a long half‐life of 53.2 ± 13.2 minutes. The clearance of ML was monoexponential, with a half‐life 7.4 ± 0.4 minutes. MR imaging revealed a pronounced uptake in bone marrow, including the iliac bone, femur, tibia, and upper and lower vertebrae. The bone marrow uptake of ML‐PEG was comparable to that of ML, with both reaching a plateau within 30 minutes following injection. Fast spin‐echo T2‐weighted imaging was found to provide optimal contrast enhancement and allowed a clear depiction of red to yellow marrow conversion due to normal aging. While the use of magnetoliposomes can provide the added benefit of therapeutic drug or gene delivery, further investigation is warranted to assess their usefulness in differentiating normal vs. abnormal marrow conditions.J. Magn. Reson. Imaging 1999;9:329–335.

Relaxometry and magnetometry of the MR contrast agent MION-46L.

MION‐46L is an ultrasmall monocrystalline superparamagnetic (SPM) iron oxide that is of current interest as an MR contrast agent. It is believed to consist primarily of small maghemite or magnetite crystals that possess a SPM moment, but the exact magnetic properties and related mechanisms of T1 and T2 proton relaxation enhancement are not well understood. We have obtained a comprehensive data set consisting of magnetization curves, EPR spectra, and 1/T1 and 1/T2 nuclear magnetic relaxation dispersion (NMRD) profiles for this contrast agent. The magnetization curves show a primary curvature consistent with a SPM moment of 10,300 Bohr magnetons (BM) per particle. In addition, there is a secondary high‐field curvature that is consistent with a smaller SPM moment of 1600 BM, which may be responsible for the observed high‐field increase in 1/T2. Finally, there appear to be a considerable number of paramagnetic ions present that are needed to account for the high‐field increase in magnetization, and that can provide an alternative explanation for the magnitude of the low‐field T1 plateau. This “three‐phase model” appears to be successful in explaining in a self‐consistent and quantitative manner the combined results of the magnetometry, relaxometry, and EPR studies. Magn Reson Med 42:379–384, 1999. Published 1999 Wiley‐Liss, Inc.

Study of relapsing remitting experimental allergic encephalomyelitis SJL mouse model using MION-46L enhanced in vivo MRI: Early histopathological correlation

MION‐46L, a superparamagnetic iron oxide contrast agent, was investigated for its ability to increase the sensitivity of in vivo 3D MRI in the detection of brain lesions in a chronic experimental allergic encephalomyelitis (crEAE) mouse model. Lesion conspicuity on postcontrast 3D MRI was dramatically enhanced as compared to precontrast images corresponding to areas of inflammatory and demyelinating lesions. MION‐46L could be detected on Prussian blue iron stain in the vascular endothelium, the perivascular space, and in macrophages within perivascular cuffs and areas of inflammation and demyelination. By taking advantage of the MION‐46L induced macroscopic susceptibility effect, acute early lesions measuring only 100 μm in diameter could be detected. MION‐46L enhanced MRI may be used to 1) provide a unique sensitivity in EAE lesion detection and correlate imaging to histopathology; 2) help to understand EAE lesion development and its underlying pathophysiology; and 3) eventually assist in preclinical screening of new experimental therapies directed at patients with multiple sclerosis (MS). J. Neurosci. Res. 52:549–558, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Magnetic resonance imaging of brain iron in health and disease

Brain iron is a major contributor to magnetic resonance imaging (MRI) contrast in normal gray matter, and its role in the pathogenesis of different neurological disorders has also become apparent. Non-heme brain iron is present in the brain mainly in the form of ferritin. The unique magnetic properties of ferritin determine different signal changes on both T1- and T2-weighted images, and the T2 relaxation rates have a linear dependence on applied field strength. This finding is typical for ferric oxyhydroxide cores. The resulting T2-shortening also depends on echo-spacing used in the imaging sequence as well as on the water diffusion coefficient and the size of the ferritin cluster. Quantitation of non-heme brain iron by MRI aids in the diagnosis and monitoring of different neurological diseases.

Dynamic relaxometry: application to iron uptake by ferritin

Abstractu2002We introduce dynamic relaxometry as a novel technique for studying biochemical reactions, such as those leading to mineral formation (biomineralization). This technique was applied to follow the time course of iron oxidation and hydrolysis by the protein ferritin. Horse spleen apoferritin was loaded with single additions of 4, 10, 20, 40, and 100 ferrous ions per protein, and with multiple additions of 4, 10, 20, and 100 ferrous ions. The NMR T2 relaxation time was then measured sequentially and continuously for up to 24 h. At low loading factors of 4–10 Fe atoms/molecule, the iron is rapidly bound and oxidized by the protein on a time scale of approximately 15u2009s to several minutes. At intermediate loading factors (10–40), rapid initial oxidation was observed, followed by the formation of antiferromagnetic clusters. This process occurred at a much slower rate and continued for up to several hours, but was inhibited at lower pH values. At higher loading factors (40–1000), iron oxidation may occur directly on the core, and this process may continue for up to 24 h following the initial loading. Dynamic relaxometry appears to be a potentially powerful technique that may well have applications beyond the study of iron upake by the ferritin protein.