Characterization of estrogen‐receptor‐targeted contrast agents in solution,breast cancer cells,and tumors in vivo

The estrogen receptor (ER) is a major prognostic biomarker of breast cancer, currently determined in surgical specimens by immunohistochemistry. Two new ER‐targeted probes, pyridine‐tetra‐acetate‐Gd chelate (PTA‐Gd) conjugated either to 17β‐estradiol (EPTA‐Gd) or to tamoxifen (TPTA‐Gd), were explored as contrast agents for molecular imaging of ER. In solution, both probes exhibited a micromolar ER binding affinity, fast water exchange rate (～10⁷ s^?1), and water proton‐relaxivity of 4.7–6.8 mM^?1 s^?1. In human breast cancer cells, both probes acted as estrogen agonists and enhanced the water protons T₁ relaxation rate and relaxivity in ER‐positive as compared to ER‐negative cells, with EPTA‐Gd showing a higher ER‐specific relaxivity than TPTA‐Gd. In studies of breast cancer tumors in vivo, EPTA‐Gd induced the highest enhancement in ER‐positive tumors as compared to ER‐negative tumors and muscle tissue, enabling in vivo detection of ER. TPTA‐Gd demonstrated the highest enhancement in muscle tissue indicating nonspecific interaction of this agent with muscle components. The extracellular contrast agents, PTA‐Gd and GdDTPA, showed no difference in the perfusion capacity of ER‐positive and ‐negative tumors confirming the specific interaction of EPTA‐Gd with ER. These findings lay a basis for the molecular imaging of the ER using EPTA‐Gd as a template for further developments. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

In vivo MRI visualization of different cell populations labeled with PARACEST agents

Conventional T₁‐ or T₂‐MRI contrast agents do not allow to track the distribution of different cell populations simultaneously because the effects of relaxation enhancers are additive. Herein, it is shown that paramagnetic chemical exchange saturation transfer agents offer the opportunity to visualize different cell populations in vitro and in vivo by ¹H‐MRI. Yb‐ and Eu‐HPDO3A complexes have been used to label murine macrophages (J774.A1) and melanoma cells (B16‐F10), respectively. By selective irradiation of the highly‐shifted OH resonances of the two chemical exchange saturation transfer agents, it has been shown that tracking of the two cell types is possible. These PARAmagnetic Chemical Exchange Saturation Transfer agents have a tremendous potential for clinical translation as they share the same stability and in vivo pharmacokinetic properties of Gd‐HPDO3A (ProHance^®), which is a widely used clinically approved MRI agent. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

T2*‐relaxivity contrast imaging: First results

In this study, T₂*‐ relaxivity contrast imaging (RCI) is proposed for new contrast generation in MRI. The method produces images of relaxivities r*_2,vasc and r*_2,EES caused by susceptibility gradients across the vessel walls and cell membranes, respectively. The sensitivity to noise was assessed with a simulation study, and initial results are presented for five colorectal tumor xenografts in nude mice. Simulations show that the new relaxivity parameters are at least as accurate and precise as standard parameters such as plasma volume and interstitial volume. Mean values of both relaxivities were significantly different (r*_2,vasc = 10.9 ± 2.9 mM^?1 s^?1 and r*_2,EES = 15.6 ± 2.6 mM^?1 s^?1). r*_2,vasc (r = 0.67) and r*_2,EES (r = 0.52) were weakly correlated with plasma volume and interstitial volume, respectively. Images of r*_2,vasc and r*_2,EES reveal a different tumor structure than plasma volume and interstitial volume maps. These results suggest that relaxivity contrast imaging is practically feasible and might offer supplementary information compared to dynamic contrast‐enhanced‐MRI. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

T2 exchange agents: A new class of paramagnetic MRI contrast agent that shortens water T2 by chemical exchange rather than relaxation

Exchange of water molecules between the frequency‐shifted inner‐sphere of a paramagnetic lanthanide ion and aqueous solvent can shorten the T₂ of bulk water protons. The magnitude of the line‐broadening T₂ exchange (T_2exch) is determined by the lanthanide concentration, the chemical shift of the exchanging water molecule, and the rate of water exchange between the two pools. A large T_2exch contribution to the water linewidth was initially observed in experiments involving Eu³⁺‐based paramagnetic chemical exchange saturation transfer agents in vivo at 9.4 T. Further in vitro and in vivo experiments using six different Eu³⁺ complexes having water exchange rates ranging from zero (no exchange) to 5 × 10⁶ s^?1 (fast exchange) were performed. The results showed that the exchange relaxivity (r_2exch) is small for complexes having either very fast or very slow exchange, but reaches a well‐defined maximum for complexes with intermediate water exchange rates. These experimental results were verified by Bloch simulations for two site exchange. This new class of T_2exch agent could prove useful in the design of responsive MRI contrast agents for molecular imaging of biological processes. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

Overcoming the low relaxivity of gadofosveset at high field with spin locking

The contrast agent gadofosveset, which binds reversibly to serum albumin, has a high longitudinal relaxivity at lower magnetic fields (≤3.0 T) but a much lower relaxivity at high fields. Spin locking is sensitive to macromolecular content; it is hypothesized that combining this technique with the albumin‐binding properties of gadofosveset may enable increased relaxivity at high fields. In vitro measurements at 4.7 T found significantly higher spin‐lock relaxation rates, R_1ρ (1/T_1ρ), when gadofosveset was serum albumin‐bound than when unbound. R_1ρ values for a nonbinding contrast agent (gadopentetate dimeglumine) in serum albumin were similar to those for unbound gadofosveset. R₂ (1/T₂) values were also significantly higher at 4.7 T for serum albumin‐bound gadofosveset than for unbound. Spin locking at high field generates significantly higher relaxation rates for gadofosveset than conventional contrast agents and may provide a method for differentiating free and bound molecules at these field strengths. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.     

Enhanced positive‐contrast visualization of paramagnetic contrast agents using phase images

Iron oxide–based MRI contrast agents are increasingly being used to noninvasively track cells, target molecular epitopes, and monitor gene expression in vivo. Detecting regions of contrast agent accumulation can be challenging if resulting contrast is subtle relative to endogenous tissue hypointensities. A postprocessing method is presented that yields enhanced positive‐contrast images from the phase map associated with T₂*‐weighted MRI data. As examples, the method was applied to an agarose gel phantom doped with superparamagnetic iron‐oxide nanoparticles and in vivo and ex vivo mouse brains inoculated with recombinant viruses delivering transgenes that induce overexpression of paramagnetic ferritin. Overall, this approach generates images that exhibit a 1‐ to 8‐fold improvement in contrast‐to‐noise ratio in regions where paramagnetic agents are present compared to conventional magnitude images. This approach can be used in conjunction with conventional T₂* pulse sequences, requires no prescans or increased scan time, and can be applied retrospectively to previously acquired data. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

A human ferritin iron oxide nano‐composite magnetic resonance contrast agent

Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)‐iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R₂ relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn‐iron oxide contrast agent to assess inflammatory disease status such as macrophage‐rich atherosclerotic plaques in vivo. Magn Reson Med 60:1073–1081, 2008. © 2008 Wiley‐Liss, Inc.     

Convertible manganese contrast for molecular and cellular MRI

We describe here the use of inorganic manganese based particles as convertible MRI agents. As has been demonstrated with iron oxide particles, manganese oxide and manganese carbonate particles can be internalized within phagocytotic cells, being subsequently shuttled to endosomes and/or lysosomes. As intact particles, only susceptibility‐induced MRI contrast is exhibited, most often seen as dark contrast in susceptibility‐weighted images. Modulation of MRI contrast is accomplished by the selective degradation of these particles within the endosomal and lysosomal compartments of cells. Upon particle deconstruction in the endosomes and lysosomes, the dissolved Mn²⁺ acts as a T₁ agent, eliciting bright contrast in T₁‐weighted images. This modulation of MRI contrast is demonstrated both in vitro in cells in culture, and also in vivo, in rat brain. These particles are the potential building blocks for an entire class of new environmentally responsive MRI contrast agents. Magn Reson Med 60:265–269, 2008. © 2008 Wiley‐Liss, Inc.     

Cellular compartmentalization of internalized paramagnetic liposomes strongly influences both T1 and T2 relaxivity

In recent years, numerous Gd³⁺‐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 α_νβ₃ integrin. The goal of this study was to investigate the consequences of this amplification approach for the T₁‐ and T₂‐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 α_νβ₃ integrin receptor. Non‐targeted liposomes served as a control. This study showed that α_νβ₃ 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. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

ΔR2* gadolinium‐diethylenetriaminepentacetic acid relaxivity in venous blood

The accuracy of perfusion measurements using dynamic, susceptibility‐weighted, contrast‐enhanced MRI depends on estimating contrast agent concentration in an artery, i.e., the arterial input function. One of the difficulties associated with obtaining an arterial input function are partial volume effects when both blood and brain parenchyma occupy the same pixel. Previous studies have attempted to correct arterial input functions which suffer from partial volume effects using contrast concentration in venous blood. However, the relationship between relaxation and concentration (C) in venous blood has not been determined in vivo. In this note, a previously employed fitting approach is used to determine venous relaxivity in vivo. In vivo relaxivity is compared with venous relaxivity measured in vitro in bulk blood. The results show that the fitting approach produces relaxivity calibration curves which give excellent agreement with arterial measurements. Magn Reson Med 69:1104–1108, 2013. © 2012 Wiley Periodicals, Inc.     

High‐contrast in vivo visualization of microvessels using novel FeCo/GC magnetic nanocrystals

FeCo‐graphitic carbon shell nanocrystals are a novel MRI contrast agent with unprecedented high per‐metal‐atom‐basis relaxivity (r₁ = 97 mM^‐1 sec^‐1, r₂ = 400 mM^‐1 sec^‐1) and multifunctional capabilities. While the conventional gadolinium‐based contrast‐enhanced angiographic magnetic MRI has proven useful for diagnosis of vascular diseases, its short circulation time and relatively low sensitivity render high‐resolution MRI of morphologically small vascular structures such as those involved in collateral, arteriogenic, and angiogenic vessel formation challenging. Here, by combining FeCo‐graphitic carbon shell nanocrystals with high‐resolution MRI technique, we demonstrate that such microvessels down to ～100 μm can be monitored in high contrast and noninvasively using a conventional 1.5‐T clinical MRI system, achieving a diagnostic imaging standard approximating that of the more invasive X‐ray angiography. Preliminary in vitro and in vivo toxicity study results also show no sign of toxicity. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Simplified synthesis and relaxometry of magnetoferritin for magnetic resonance imaging

Magnetoferritin nanoparticles have been developed as high‐relaxivity, functional contrast agents for MRI. Several previous techniques have relied on unloading native ferritin and re‐incorporation of iron into the core, often resulting in a polydisperse sample. Here, a simplified technique is developed using commercially available horse spleen apoferritin to create monodisperse magnetoferritin. Iron oxide atoms were incorporated into the protein core via a step‐wise Fe(II)Chloride addition to the protein solution under low O₂ conditions; subsequent filtration steps allow for separation of completely filled and superparamagnetic magnetoferritin from the partially filled ferritin. This method yields a monodisperse and homogenous solution of spherical particles with magnetic properties that can be used for molecular magnetic resonance imaging. With a transverse per‐iron and per‐particle relaxivity of 78 mM^?1 sec^?1 and 404,045 mM^?1 sec^?1, respectively, it is possible to detect ～10 nM nanoparticle concentrations in vivo. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Optimization of iron oxide nanoparticle detection using ultrashort echo time pulse sequences: Comparison of T1, T2*, and synergistic T1 − T2* contrast mechanisms

Iron oxide nanoparticles (IONPs) are used in various MRI applications as negative contrast agents. A major challenge is to distinguish regions of signal void due to IONPs from those due to low signal tissues or susceptibility artifacts. To overcome this limitation, several positive contrast strategies have been proposed. Relying on IONP T₁ shortening effects to generate positive contrast is a particularly appealing strategy because it should provide additional specificity when associated with the usual negative contrast from effective transverse relaxation time (T₂*) effects. In this article, ultrashort echo time imaging is shown to be a powerful technique which can take full advantage of both contrast mechanisms. Methods of comparing T₁ and T₂* contrast efficiency are described and general rules that allow optimizing IONP detection sensitivity are derived. Contrary to conventional wisdom, optimizing T₁ contrast is often a good strategy for imaging IONPs. Under certain conditions, subtraction of a later echo signal from the ultrashort echo time signal not only improves IONP specificity by providing long T₂* background suppression but also increases detection sensitivity, as it enables a synergistic combination of usually antagonist T₁ and T₂* contrasts. In vitro experiments support our theory, and a molecular imaging application is demonstrated using tumor‐targeted IONPs in vivo. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Polydisulfide manganese(II) complexes as non-gadolinium biodegradable macromolecular MRI contrast agents

Purpose:

To develop safe and effective manganese(II) ‐based biodegradable macromolecular MRI contrast agents.

Materials and Methods:

In this study, we synthesized and characterized two polydisulfide manganese(II) complexes, Mn‐DTPA cystamine copolymers and Mn‐EDTA cystamine copolymers, as new biodegradable macromolecular MRI contrast agents. The contrast enhancement of the two manganese‐based contrast agents were evaluated in mice bearing MDA‐MB‐231 human breast carcinoma xenografts, in comparison with MnCl₂.

Results:

The T₁ and T₂ relaxivities were 4.74 and 10.38 mM^?1s^?1 per manganese at 3T for Mn‐DTPA cystamine copolymers (M_n = 30.50 kDa) and 6.41 and 9.72 mM^?1s^?1 for Mn‐EDTA cystamine copolymers (M_n = 61.80 kDa). Both polydisulfide Mn(II) complexes showed significant liver, myocardium and tumor enhancement.

Conclusion:

The manganese‐based polydisulfide contrast agents have a potential to be developed as alternative non‐gadolinium contrast agents for MR cancer and myocardium imaging. J. Magn. Reson. Imaging 2012;35:737‐744. © 2011 Wiley Periodicals, Inc.

     

lacZ as a genetic reporter for real‐time MRI

Molecular imaging based on MRI is currently hampered by the lack of genetic reporters for in vivo imaging. We determined that the commercially available substrate S‐Gal? can be used to detect genetically engineered β‐galactosidase expressing cells by MRI. The effect and specificity of the reaction between β‐galactosidase and S‐Gal? on MRI contrast were determined both in vitro and in vivo. β‐galactosidase activity in the presence of S‐Gal? resulted in enhanced T₂ and T*₂ MR‐contrast, which was amplified with increasing magnetic field strengths (4.7‐17.6 T) in phantom studies. Using both lacZ⁺ transgenic animals and lacZ⁺ tissue transplants, we were able to detect labeled cells in live animals in real time. Similar to phantom studies, detection of the labeled cells/tissues in vivo was enhanced at high magnetic fields. These results demonstrate that the genetic reporter, lacZ, can be used as an in vivo marker gene using high‐field‐strength MRI. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Protein polymer MRI contrast agents: Longitudinal analysis of biomaterials in vivo

Despite recent advances in tissue engineering to regenerate biological function by combining cells with material supports, development is hindered by inadequate techniques for characterizing biomaterials in vivo. Magnetic resonance imaging is a tomographic technique with high temporal and spatial resolution and represents an excellent imaging modality for longitudinal noninvasive assessment of biomaterials in vivo. To distinguish biomaterials from surrounding tissues for magnetic resonance imaging, protein polymer contrast agents were developed and incorporated into hydrogels. In vitro and in vivo images of protein polymer hydrogels, with and without covalently incorporated protein polymer contrast agents, were acquired by magnetic resonance imaging. T₁ values of the labeled gels were consistently lower when protein polymer contrast agents were included. As a result, the protein polymer contrast agent hydrogels facilitated fate tracking, quantification of degradation, and detection of immune response in vivo. For the duration of the in vivo study, the protein polymer contrast agent‐containing hydrogels could be distinguished from adjacent tissues and from the foreign body response surrounding the gels. The hydrogels containing protein polymer contrast agent have a contrast‐to‐noise ratio 2‐fold greater than hydrogels without protein polymer contrast agent. In the absence of the protein polymer contrast agent, hydrogels cannot be distinguished by the end of the gel lifetime. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Kinetics of avidin‐induced clearance of biotinylated bimodal liposomes for improved MR molecular imaging

Dual labeled liposomes, carrying both paramagnetic and fluorescent lipids, were recently proposed as potent contrast agents for MR molecular imaging. These nanoparticles are coated with poly(ethylene glycol) (PEG) to increase their blood circulation half‐life, which should allow extensive accumulation at the targeted site. To eliminate nonspecific blood pool signal from the MR images, the circulating liposomes should ideally be cleared from the circulation when sufficient target‐specific contrast enhancement is obtained. To that aim, we designed an avidin chase that allowed controlled and rapid clearance of paramagnetic biotinylated liposomes from the blood circulation in C57BL/6 mice. Avidin‐induced alterations in blood clearance kinetics and tissue distribution were studied quantitatively by determination of the Gd content in blood and tissue samples ex vivo. Intrinsic liposomal blood clearance showed bi‐exponential behavior with half‐lives t_1/2α = 2.1 ± 1.1 and t_1/2β = 15.1 ± 5.4 hours, respectively. In contrast, the contrast agent was cleared from the blood by the avidin infusion to <1% of the initial dose within 4 hours. Avidin‐induced liposomal blood clearance was also demonstrated in vivo by dynamic T₁‐weighted MRI. The ability to rapidly clear circulating contrast agents opens up exciting possibilities to study targeting kinetics, to increase the specificity of molecular MRI and to optimize nanoparticulate contrast agent formulations. Magn Reson Med 60:1444–1456, 2008. © 2008 Wiley‐Liss, Inc.     

Combined off-resonance imaging and T2 relaxation in the rotating frame for positive contrast MR imaging of infection in a murine burn model

Purpose

To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages in inflammation and infection. Positive‐contrast MR imaging provides an alternative to negative‐contrast MRI, exploiting the chemical shift induced by ultra‐small superparamagnetic iron‐oxide (USPIO) nanoparticles to nearby water molecules. We introduce a novel combination of off‐resonance (ORI) positive‐contrast MRI and T_2ρ relaxation in the rotating frame (ORI‐T_2ρ) for positive‐contrast MR imaging of USPIO.

Materials and Methods

We tested ORI‐T_2ρ in phantoms and imaged in vivo the accumulation of USPIO‐labeled macrophages at the infection site in a mouse model of burn trauma and infection with Pseudomonas aeruginosa (PA). PA infection is clinically important. The USPIO nanoparticles were injected directly in the animals in solution, and macrophage labeling occurred in vivo in the animal model.

Results

We observed a significant difference between ORI‐T_2ρ and ORI, which leads us to suggest that ORI‐T_2ρ is more sensitive in detecting USPIO signal. To this end, the ORI‐T_2ρ positive contrast method may prove to be of higher utility in future research.

Conclusion

Our results may have direct implications in the longitudinal monitoring of infection, and open perspectives for testing novel anti‐infective compounds. J. Magn. Reson. Imaging 2010;32:1172–1183. © 2010 Wiley‐Liss, Inc.     

Magnetic resonance imaging contrast agents: Overview and perspectives

Magnetic resonance imaging (MRI) is a non-invasive clinical imaging modality, which has become widely used in the diagnosis and/or staging of human diseases around the world. Some MRI examinations include the use of contrast agents. The categorizations of currently available contrast agents have been described according to their effect on the image, magnetic behavior and biodistribution in the body, respectively. In this field, superparamagnetic iron oxide particles and soluble paramagnetic metal chelates are two main classes of contrast agents for MRI. This review outlines the research and development of MRI contrast agents. In future, the ideal MRI contrast agent will be focused on the neutral tissue- or organ-targeting materials with high relaxivity and specificity, low toxicity and side effects, suitable long intravascular duration and excretion time, high contrast enhancement with low dose in vivo, and with minimal cost.     

Reproducibility and correlation between quantitative and semiquantitative dynamic and intrinsic susceptibility‐weighted MRI parameters in the benign and malignant human prostate

Purpose:

To assess the reproducibility of relaxivity‐ and susceptibility‐based dynamic contrast‐enhanced magnetic resonance imaging (MRI) in the benign and malignant prostate gland and to correlate the kinetic parameters obtained.

Materials and Methods:

Twenty patients with prostate cancer underwent paired scans before and after androgen deprivation therapy. Quantitative parametric maps for T₁‐ and T₂*‐weighted parameters were calculated (K^trans, k_ep,v_e, IAUC₆₀, rBV, rBF, and R₂*). The reproducibility of and correlation between each parameter were determined using standard methods at both timepoints.

Results:

T₁‐derived parameters are more reproducible than T₂*‐weighted measures, both becoming more variable following androgen deprivation (variance coefficients for prostate K^trans and rBF increased from 13.9%–15.8% and 42.5%–90.8%, respectively). Tumor R₂* reproducibility improved after androgen ablation (23.3%–11.8%). IAUC₆₀ correlated strongly with K^trans, v_e, and k_ep (all P < 0.001). R₂* did not correlate with other parameters.

Conclusion:

This study is the first to document the variability and repeatability of T₁‐ and T₂*‐weighted dynamic MRI and intrinsic susceptibility‐weighted MRI for the various regions of the human prostate gland before and after androgen deprivation. These data provide a valuable source of reference for groups that plan to use dynamic contrast‐enhanced MRI or intrinsic susceptibility‐weighted MRI for the assessment of treatment response in the benign or malignant prostate. J. Magn. Reson. Imaging 2010;32:155–164. © 2010 Wiley‐Liss, Inc.