Daniela Delli Castelli

Challenges for Molecular Magnetic Resonance Imaging

3.3. Magnetic Particle Imaging 3029 4. Challenges for CEST Agents 3029 4.1. Technical Issues 3029 4.2. Chemical Issues 3031 4.3. Biological Issues 3032 5. Challenges for Heteronuclear MR Imaging 3033 5.1. F-Based Probes 3033 6. Challenges for Hyperpolarized Probes 3034 6.1. Brute Force 3034 6.2. Optical Pumping and Spin Exchange 3035 6.3. Dynamic Nuclear Polarization (DNP) 3035 6.4. para-Hydrogen Induced Polarization (PHIP) 3037 6.5. Use of Gd Contrast Agents with Hyperpolarized Substances 3038

Paramagnetic lanthanide(III) complexes as pH-sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications

The recently introduced new class of contrast agents (CAs) based on chemical exchange saturation transfer (CEST) may have a huge potential for the development of novel applications in the field of MRI. In this work we explored the CEST properties of a series of Lanthanide(III) complexes (Ln = Eu, Dy, Ho, Er, Tm, Yb) with the macrocyclic DOTAM‐Gly ligand, which is the tetraglycineamide derivative of DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). These complexes possess two pools of exchangeable protons represented by the coordinated water and the amide protons. Yb‐DOTAM‐Gly displays the most interesting CEST properties when its amide N‐H resonance (16 ppm upfield H2O signal) is irradiated. Up to 70% suppression of the water signal is obtained at pH 8. As the exchange rate of amide protons is base‐catalyzed, Yb‐DOTAM‐Gly results to be an efficient pH‐responsive probe in the 5.5–8.1 pH range. Moreover, a ratiometric method has been set up in order to remove the dependence of the observed pH responsiveness from the absolute concentration of the paramagnetic agent. In fact, the use of a mixture of Eu‐DOTAM‐Gly and Yb‐DOTAM‐Gly, whose exchangeable proton pools are represented by the coordinated water (ca. 40 ppm downfield H2O signal at 312K) and amide protons, respectively, produces a pH‐dependent CEST effect which is the function of the concentration ratio of the two complexes. Magn Reson Med 47:639–648, 2002.

Encoding the frequency dependence in MRI contrast media: the emerging class of CEST agents.

CEST agents represent a very promising class of MRI contrast media as they encode a frequency dependence that is not like the classical relaxation-based agents. This peculiar property enables novel applications such as the detection of more than one agent in the same MR image as well as the set-up of ratiometric methods for the quantitative assessment of physico-chemical and biological parameters that characterize the micro-environment in which they are distributed. This survey is aimed at providing the reader with the basic properties and the potential of these compounds. Fundamental aspects, such as the theoretical basis of the saturation transfer via chemical exchange, the generation of the CEST contrast, the classification and sensitivity of CEST agents, and some representative examples displaying their potential in the field of MR-molecular imaging, are presented and discussed in detail.

Ln(III)-DOTAMGly complexes: a versatile series to assess the determinants of the efficacy of paramagnetic chemical exchange saturation transfer agents for magnetic resonance imaging applications.

Rationale and Objectives:Paramagnetic Ln-DOTAMGly complexes (Ln ≠ La, Lu, and Gd) are the prototypes of a novel class of contrast agents for magnetic resonance imaging based on chemical exchange saturation transfer (CEST). Their ability to reduce the water signal intensity depends on the interplay of several physico-chemical properties of the agent and instrumental parameters. This study aims to identify possible routes for their optimization Methods:Saturation transfer (ST) has been measured in vitro at 7.05 T as a function of pH, temperature, and concentration of the agent. Results:Large saturation transfer effects have been observed upon irradiating the coordinated water protons (for Ln = Pr, Nd, Eu, and Tb). The comparison of the results obtained by irradiating water versus amide protons allows the set-up of ratiometric methods through which the ST response can be made independent on the concentration of the agent. Conclusions:The modulation of the magnetic properties along the lanthanide series allows an in-depth understanding of the determinants of ST effect and provides useful insights for the design of more efficient agents.

YbIII‐HPDO3A: A Dual pH‐ and Temperature‐Responsive CEST Agent

The use of contrast agents (CAs) has been introduced about 25 years ago. Since then, a number of paramagnetic gadolinium(III) complexes have been proposed to tackle relevant bio-medical issues by endowing the systems with targeting and responsive capabilities. Responsive agents suffer from the drawback that the exploitation of the responsiveness of their relaxivity towards given physicochemical parameters requires the knowledge of the actual concentration of the agent in the region of interest. This drawback may be overcome by exploiting the frequencyencoding contrast that can be generated by chemical exchange saturation transfer (CEST) agents. In fact, it has been demonstrated that such probes may allow the detection of a concentration independent magnetic resonance imaging (MRI) response by means of ratiometric approaches. The latter require the design of agents provided with two pools of exchanging protons that show a different dependence of their CEST properties (e.g. exchange rate or chemical shift) toward the pH value of interest. Alterations of the pH values are involved in several pathological states, and, consequently, there is a high demand for developing in vivo protocols to monitor pH values. Within the field of CEST agents, this task has been so far addressed by using either diamagnetic or paramagnetic agents. The use of the latter class of agents (ParaCEST) is particularly useful because the larger chemical shift separation present in paramagnetic complexes allows the exploration of faster proton-exchange rates, that, in turn, may result into a marked sensitivity enhancement. ParaCEST agents have also been investigated as temperature reporters that exploit the effects of the exchange rates or chemical shift [11] of the mobile protons, and the overall stability of the agents. As it might be of interest (e.g. for imaging-guided hyperthermia treatments) to assess simultaneously pH and temperature, it has been deemed of relevance to merge the two responses into the same compound. To select a system with a good potential for a clinical translation, the choice has been directed toward LnHPDO3A complexes (HPDO3A = 10-(2-hydroxypropyl)1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) in virtue of the successful clinical use of Gd-HPDO3A (ProHance). Thus, Ln-HPDO3A complexes should have thermodynamic and kinetic stabilities, in vivo distribution, and excretion properties very similar to those shown by ProHance. Recently, it has been demonstrated that hydroxylic protons can be successfully used for generating CEST contrast when they are close enough to the paramagnetic center. 14] Hence, as the exchange rate of such protons is expected to be pH dependent, these chelates may act as pH sensitive ParaCEST agents. Concerning the choice of the lanthanide, it has been reported that, when the mobile pool is different from metal coordinated water protons, Yb is the most efficient ion because of to the good compromise between shifting ability and T2 shortening. [10,15] Hence, the Yb-HPDO3A complex was selected for achieving the purpose of this work. The H NMR spectrum of this complex (Figure 1) indicates the presence in solution of two species that strongly differ in the line width of their resonances.

Determination of water permeability of paramagnetic liposomes of interest in MRI field.

The water permeability of various liposome membranes has been determined at 298K by measuring the NMR longitudinal water proton relaxation rate of vesicles encapsulating the clinically approved Gd-HPDO3A complex (HPDO3A=10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). Two basic formulations based on DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyl-oleylphosphatidylcholine) phospholipids were selected and investigated. Furthermore, the permeability changes caused by the membrane incorporation of amphiphiles like cholesterol and/or metal complexes of interest for designing improved liposome-based MRI contrast agents, were also investigated. The incorporation of cholesterol and metal complexes bearing C18 saturated chains in POPC-based liposomes reduces the water diffusivity across the membrane bilayer. On the contrary, the incorporation of a macrocyclic metal complex bearing four C12 alkylic chains, one for each coordination arm of the ligand, considerably enhances the water permeability in DPPC-based liposomes. Finally, it is reported that the permeability of POPC-based bilayer is increased when the liposomes are subjected to an osmotic stress.

Paramagnetic liposomes as innovative contrast agents for magnetic resonance (MR) molecular imaging applications.

This article illustrates some innovative applications of liposomes loaded with paramagnetic lanthanide‐based complexes in MR molecular imaging field. When a relatively high amount of a GdIII chelate is encapsulated in the vesicle, the nanosystem can simultaneously affect both the longitudinal (R1) and the transverse (R2) relaxation rate of the bulk H2O H‐atoms, and this finding can be exploited to design improved thermosensitive liposomes whose MRI response is not longer dependent on the concentration of the probe. The observation that the liposome compartmentalization of a paramagnetic LnIII complex induce a significant R2 enhancement, primarily caused by magnetic susceptibility effects, prompted us to test the potential of such agents in cell‐targeting MR experiments. The results obtained indicated that these nanoprobes may have a great potential for the MR visualization of cellular targets (like the glutamine membrane transporters) overexpressing in tumor cells. Liposomes loaded with paramagnetic complexes acting as NMR shift reagents have been recently proposed as highly sensitive CEST MRI agents. The main peculiarity of CEST probes is to allow the MR visualization of different agents present in the same region of interest, and this article provides an illustrative example of the in vivo potential of liposome‐based CEST agents.

In vivo MRI multicontrast kinetic analysis of the uptake and intracellular trafficking of paramagnetically labeled liposomes

This work aims at developing a MRI method that allows to get more insight into the understanding of the in vivo fate of liposomes and their payload. The method relies on the temporal assessment of the contrast changes induced by the presence of a classical relaxation agent versus the effect induced by a CEST (chemical exchange saturation transfer) agent. Liposomes were loaded with the paramagnetic complexes, Gd-HPDO3A and [Tm-DOTMA](-) [Na](+), in order to endow the nanovesicles with the characteristic properties of T(1)/T(2) and CEST/T(2) MRI agents, respectively. The paramagnetically loaded liposomes were injected directly into the tumor (B16 melanoma xenograft in mice) where they generate T(1), T(2), and CEST MR contrasts that were quantitatively monitored over time (0-48h). The kinetic of each contrast enhancement reports about peculiar properties relative to the fate of the liposomes in the tumor environment. A kinetic model has been set-up to fit the experimental multicontrast data in order to extract the relevant information about the cellular uptake of the liposomes and the release of their payload. Upon comparing conventional stealth liposomes with pH-sensitive ones, it has been shown that the latter ones differ essentially in the step associated with the release of the drug that is likely occurring in the endosomal acidic vesicles.

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.

Methods for an improved detection of the MRI-CEST effect

CEST imaging is a recently introduced MRI contrast modality based on the use of endogenous or exogenous molecules whose exchangeable proton pools transfer saturated magnetization to bulk water, thus creating negative contrast. One of the critical issues for further development of these agents is represented by their limited sensitivity in vivo. The aim of this work is to improve the detection of CEST agents by exploring new approaches through which the saturation transfer (ST) effect can be enhanced. The performance of the proposed methods has been tested in vitro and in vivo using highly sensitive and highly shifted lipoCEST agents, and the results were compared with the standard ST evaluation mode. The acquired Z-spectra were interpolated locally and voxel-by-voxel by smoothing splines. Besides expressing the ST in the standard mode, we explore two methods, enhanced and integral ST, which better exploit all the information contained in the Z-spectrum. By combining different modes for ST assessment a significant improvement in the detection of the lipoCEST agents, both in vitro and in vivo, has been found. The results obtained from the application of the proposed methods outline the importance of post-processing analysis for highlighting the CEST-MRI contrast.