Toward quantification of hypoxia using fluorinated EuII/III-containing ratiometric probes.

Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using EuII-based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and EuII. Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated EuII/III-containing probes. We studied three different EuII/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time (T1) and 19F signal of solutions containing different ratios of EuII- and EuIII-containing complexes was plotted against the percentage of EuII-containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.

Outersphere Approach to Increasing the Persistance of Oxygen-Sensitive Europium(II)-Containing Contrast Agents for Magnetic Resonance Imaging with Perfluorocarbon Nanoemulsions toward Imaging of Hypoxia.

Radiographic mapping of hypoxia is needed to study a wide range of diseases. Complexes of Eu(II) are a promising class of molecules to fit this need, but they are generally limited by their rapid oxidation rates in vivo. Here, a perfluorocarbon-nanoemulsion perfused with N2 , forms an interface with aqueous layers to hinder oxidation of a new perfluorocarbon-soluble complex of Eu(II). Conversion of the perfluorocarbon solution of Eu(II) into nanoemulsions results in observable differences between reduced and oxidized forms by magnetic resonance imaging both in vitro and in vivo. Oxidation in vivo occurrs over a period of ≈30 min compared to <5 min for a comparable Eu(II)-containing complex without nanoparticle interfaces. These results represent a critical step toward delivery of Eu(II)-containing complexes in vivo for the study of hypoxia.

Systemic Delivery of Divalent Europium from Ligand Screening with Implications to Direct Imaging of Hypoxia.

Hypoxia is a hallmark of many diseases, including cancer, arthritis, heart and kidney diseases, and diabetes, and it is often associated with disease aggressiveness and poor prognosis. Consequently, there is a critical need for imaging hypoxia in a noninvasive and direct way to diagnose, stage, and monitor the treatment and development of new therapies for these diseases. Eu-containing contrast agents for magnetic resonance imaging have demonstrated potential for in vivo imaging of hypoxia via changes in metal oxidation state from +2 to +3, but rapid oxidation in blood limits EuII-containing complexes to studies compatible with direct injection to sites. Here, we report a new EuII-containing complex that persists in oxygenated environments and is capable of persisting in blood long enough for imaging by magnetic resonance imaging. We describe the screening of a library of ligands that led to the discovery of the complex as well as a pH-dependent mechanism that hinders oxidation to enable usefulness in vivo. These studies of the first divalent lanthanide complex that persists in oxygenated solutions open the door to the use of EuII-based contrast agents for imaging hypoxia in a wide range of diseases.

A Mouse Holder for Awake Functional Imaging in Unanesthetized Mice: Applications in 31P Spectroscopy, Manganese-Enhanced Magnetic Resonance Imaging Studies, and Resting-State Functional Magnetic Resonance Imaging.

Anesthesia is often used in preclinical imaging studies that incorporate mouse or rat models. However, multiple reports indicate that anesthesia has significant physiological impacts. Thus, there has been great interest in performing imaging studies in awake, unanesthetized animals to obtain accurate results without the confounding physiological effects of anesthesia. Here, we describe a newly designed mouse holder that is interfaceable with existing MRI systems and enables awake in vivo mouse imaging. This holder significantly reduces head movement of the awake animal compared to previously designed holders and allows for the acquisition of improved anatomical images. In addition to applications in anatomical T2-weighted magnetic resonance imaging (MRI), we also describe applications in acquiring 31P spectra, manganese-enhanced magnetic resonance imaging (MEMRI) transport rates and resting-state functional magnetic resonance imaging (rs-fMRI) in awake animals and describe a successful conditioning paradigm for awake imaging. These data demonstrate significant differences in 31P spectra, MEMRI transport rates, and rs-fMRI connectivity between anesthetized and awake animals, emphasizing the importance of performing functional studies in unanesthetized animals. Furthermore, these studies demonstrate that the mouse holder presented here is easy to construct and use, compatible with standard Bruker systems for mouse imaging, and provides rigorous results in awake mice.