Giuseppe Ferrauto

In vivo maps of extracellular pH in murine melanoma by CEST-MRI.

A novel method based on the use of Yb‐HPDO3A as MRI Para‐CEST agent for in vivo pH mapping of the tumor region in a melanoma murine model is reported. This method does not require the knowledge of the concentration of the imaging agent.

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.

An MRI Method To Map Tumor Hypoxia Using Red Blood Cells Loaded with a pO2-Responsive Gd-Agent

Hypoxia is a typical hallmark of many solid tumors and often leads to therapy resistance and the development of a more aggressive cancer phenotype. Oxygen content in tissues has been evaluated using numerous different methods for several imaging modalities, but none has yet reached the required standard of spatial and temporal resolution. Magnetic Resonance Imaging (MRI) appears to be the technique of choice and several pO2-responsive probes have been designed for it over the years. In vivo translation is often hampered in Gd-relaxation agents as it is not possible to separate effects that arise from changes in local concentration from those associated with responsive properties. A novel procedure for the MRI based assessment of hypoxia is reported herein. The method relies on the combined use of Gd-DOTP- and Gd-HPDO3A-labeled red blood cells (RBCs) where the first probe acts as a vascular oxygenation-responsive agent, while the second reports the local labeled RBC concentration in a transplanted breast tumor mouse model. The MRI assessment of oxygenation state has been validated by photoacoustic imaging and ex vivo immunofluorescence. The method refines tumor staging in preclinical models and makes possible an accurate monitoring of the relationship between oxygenation and tumor growth.

Advanced cardiac chemical exchange saturation transfer (cardioCEST) MRI for in vivo cell tracking and metabolic imaging.

An improved pre‐clinical cardiac chemical exchange saturation transfer (CEST) pulse sequence (cardioCEST) was used to selectively visualize paramagnetic CEST (paraCEST)‐labeled cells following intramyocardial implantation. In addition, cardioCEST was used to examine the effect of diet‐induced obesity upon myocardial creatine CEST contrast. CEST pulse sequences were designed from standard turbo‐spin‐echo and gradient‐echo sequences, and a cardiorespiratory‐gated steady‐state cine gradient‐echo sequence. In vitro validation studies performed in phantoms composed of 20 mM Eu‐HPDO3A, 20 mM Yb‐HPDO3A, or saline demonstrated similar CEST contrast by spin‐echo and gradient‐echo pulse sequences. Skeletal myoblast cells (C2C12) were labeled with either Eu‐HPDO3A or saline using a hypotonic swelling procedure and implanted into the myocardium of C57B6/J mice. Inductively coupled plasma mass spectrometry confirmed cellular levels of Eu of 2.1 × 10−3 ng/cell in Eu‐HPDO3A‐labeled cells and 2.3 × 10−5 ng/cell in saline‐labeled cells. In vivo cardioCEST imaging of labeled cells at ±15 ppm was performed 24 h after implantation and revealed significantly elevated asymmetric magnetization transfer ratio values in regions of Eu‐HPDO3A‐labeled cells when compared with surrounding myocardium or saline‐labeled cells. We further utilized the cardioCEST pulse sequence to examine changes in myocardial creatine in response to diet‐induced obesity by acquiring pairs of cardioCEST images at ±1.8 ppm. While ventricular geometry and function were unchanged between mice fed either a high‐fat diet or a corresponding control low‐fat diet for 14 weeks, myocardial creatine CEST contrast was significantly reduced in mice fed the high‐fat diet. The selective visualization of paraCEST‐labeled cells using cardioCEST imaging can enable investigation of cell fate processes in cardioregenerative medicine, or multiplex imaging of cell survival with imaging of cardiac structure and function and additional imaging of myocardial creatine. Copyright

Simultaneous MR imaging for tissue engineering in a rat model of stroke.

In situ tissue engineering within a stroke cavity is gradually emerging as a novel therapeutic paradigm. Considering the varied lesion topology within each subject, the placement and distribution of cells within the lesion cavity is challenging. The use of multiple cell types to reconstruct damaged tissue illustrates the complexity of the process, but also highlights the challenges to provide a non-invasive assessment. The distribution of implanted cells within the lesion cavity and crucially the contribution of neural stem cells and endothelial cells to morphogenesis could be visualized simultaneously using two paramagnetic chemical exchange saturation transfer (paraCEST) agents. The development of sophisticated imaging methods is essential to guide delivery of the building blocks for in situ tissue engineering, but will also be essential to understand the dynamics of cellular interactions leading to the formation of de novo tissue.

Frequency-Encoded MRI-CEST Agents Based on Paramagnetic Liposomes/RBC Aggregates

Paramagnetic liposomes containing Dy-HPDO3A in their inner water compartment and carrying a residual positive charge on their outer surface have been electrostatically bound to the membrane of red blood cells (RBCs). These aggregates yield two chemical exchange saturation transfer (CEST) pools represented by liposomal water protons (LipoCEST) and cytoplasmatic water protons (ErythroCEST), respectively. The absorption frequencies of the two pools fall at the negative and positive side of the solvent water resonance as expected from the dipolar (LipoCEST) and BMS (bulk magnetic susceptibility) (ErythroCEST) origin of the paramagnetic induced shift of their water protons resonances, respectively. In vivo magnetic resonance imaging (MRI) shows that the liposomes/RBC aggregates report about the vascular volume whereas the residual LipoCEST effect informs about the presence of released liposomes in the region of interest (ROI). Besides being an innovative blood cell labeling for MRI, the LipoCEST/RBC aggregates provide a route to improve the circulation lifetime of the liposomes and the CEST procedure allows assessing the deassembly of the aggregates and accumulation of the liposomes in the ROI.

Gd-loaded-RBCs for the assessment of tumor vascular volume by contrast-enhanced-MRI

The assessment of the fractional vascular volume (vV) in the tumor area is of great interest in the characterization of tumor and it can be useful to monitor the outcome of anti-angiogenetic therapies. The high spatial and temporal resolution of Magnetic Resonance Imaging makes it the election imaging modality to monitor in vivo the vascular volume changes. Commonly used MRI methods to obtain this information rely on the administration of contrast agents that modify the bulk water relaxation times but, unfortunately, they can provide only an estimate of vV since they are not fully retained in the vascular space. Herein, Gd-loaded Red Blood Cells (Gd-RBCs) are proposed as a contrast agent able to provide quantitative information on tumor vascularization. Being Gd-RBCs fully retained in the vascular space, the proposed method does not suffer for the limitations associated to the use of extracellular Gd-agents that quickly extravasate in the leaky tumor vasculature. Furthermore, the long half-life and biocompatibility of Gd-RBCs allows repeating the measurement many times upon their administration; this ensures the possibility to in vivo evaluate the change of vascular volume during tumor growth. For these reasons, Gd-RBCs may represent a highly biocompatible imaging reporter of vasculature, able to quantitatively assess changes in the vascular volume in the ROI.

Insights on the Relaxation of Liposomes Encapsulating Paramagnetic Ln-Based Complexes

Purpose: To describe and quantify the different relaxation mechanisms operating in suspensions of liposomes that encapsulate paramagnetic lanthanide(III) complexes. Theory and Methods: The transverse relaxation rate of lanthanide‐loaded liposomes receives contribution from the exchange between intraliposomal and bulk water protons, and from magnetic susceptibility effects. Phospholipids vesicles encapsulating different Ln(III)‐HPDO3A complexes (Ln = Eu, Gd, or Dy) were prepared using the conventional thin film rehydration method. Relaxation times (T1, T2, and T2*) were measured at 14 Tesla (T) and 25°C. The effect of compartmentalization of the paramagnetic agent inside the liposomal cavity was evaluated by means of an IRON‐modified MRI sequence. Results: NMR measurements demonstrated that Curie spin relaxation is the dominant contribution (> 90%) to the observed transverse relaxation rate of paramagnetic liposomes. This was further confirmed by MRI that showed the ability of the liposome entrapped lanthanide complexes to generate IRON‐MRI positive contrast in a size dependent manner. Conclusion: The Curie spin relaxation mechanism is by far the principal mechanism involved in the T2 shortening of the water protons in suspension of paramagnetic liposomes at 14T. The access to IRON contrast extends the potential of such nanosystems as MRI contrast agents. Magn Reson Med 74:468–473, 2015.

Eight-Coordinate, Stable Fe(II) Complex as a Dual 19F and CEST Contrast Agent for Ratiometric pH Imaging

Accurate mapping of small changes in pH is essential to the diagnosis of diseases such as cancer. The difficulty in mapping pH accurately in vivo resides in the need for the probe to have a ratiometric response so as to be able to independently determine the concentration of the probe in the body independently from its response to pH. The complex FeII-DOTAm-F12 behaves as an MRI contrast agent with dual 19F and CEST modality. The magnitude of its CEST response is dependent both on the concentration of the complex and on the pH, with a significant increase in saturation transfer between pH 6.9 and 7.4, a pH range that is relevant to cancer diagnosis. The signal-to-noise ratio of the 19F signal of the probe, on the other hand, depends only on the concentration of the contrast agent and is independent of pH. As a result, the complex can ratiometrically map pH and accurately distinguish between pH 6.9 and 7.4. Moreover, the iron(II) complex is stable in air at room temperature and adopts a rare 8-coordinate geometry.

Sensitive MRI detection of internalized T1 contrast agents using magnetization transfer contrast.

This work addresses the possibility of using Magnetization Transfer Contrast (MTC) for an improved MRI detection of T1 relaxation agents. The need to improve the detection threshold of MRI agents is particularly stringent when the contrast agents failed to accumulate to the proper extent in targeting procedures. The herein reported approach is based on the T1 dependence of MT contrast. It has been assessed that MT contrast can allow the detection of a Gd‐containing agent at a lower detection threshold than the one accessible by acquiring T1W images. Measurements have been carried out either in TS/A cells or in vivo in a syngeneic murine breast cancer model. The reported data showed that in cellular experiments the MTC method displays a better sensitivity with respect to the common T1W experiments. In particular, the reached detection threshold allowed the visualization of samples containing only 2% of Gd‐labeled cells diluted in unlabeled cells. In vivo experiments displayed a more diversified scheme. In particular, the tumor region showed two distinct behaviors accordingly with the localization of the imaging probe. The probe located in the tumor core could be detected to the same extent either by T1w or MTC contrast. Conversely, the agent located in the tumor rim was detected with a larger sensitivity by the MTC method herein described. Copyright