Daniel Calle

Environmentally Sensitive Paramagnetic and Diamagnetic Contrast Agents for Nuclear Magnetic Resonance Imaging and Spectroscopy

Even though alterations in the microenvironmental properties of tissues underlie the development of the most prevalent and morbid pathologies, they are not directly observable in vivo by Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) methods. This circumstance has lead to the development of a wide variety of exogenous paramagnetic and diamagnetic MRI and MRS probes able to inform non invasively on microenvironmental variables such as pH, pO(2), ion concentration o even temperature. This review covers the fundamentals of environmental contrast and the current arsenal of endogenous and exogenous MRI and MRS contrast enhancing agents available to visualize it. We begin describing the physicochemical background necessary to understand paramagnetic and diamagnetic contrast enhancement with a special reference to novel magnetization transfer and (13)C hyperpolarization strategies. We describe then the main macrocyclic structures used to support the environmentally sensitive paramagnetic sensors, including CEST and PARACEST pH sensitive probes, temperature probes and enzyme activity or gene expression activatable probes. Finally we address the most commonly used diamagnetic contrast agents including imidazolic derivatives to reveal extracellular pH and tissue pO(2) values by MRS. The potential applications of these agents in multimodal and molecular imaging approaches are discussed.

Magnetoliposomes Loaded with Poly-Unsaturated Fatty Acids as Novel Theranostic Anti-Inflammatory Formulations

We describe the preparation, physico-chemical characterization and anti-inflammatory properties of liposomes containing the superparamagnetic nanoparticle Nanotex, the fluorescent dye Rhodamine-100 and omega-3 polyunsaturated fatty acid ethyl ester (ω-3 PUFA-EE), as theranostic anti-inflammatory agents. Liposomes were prepared after drying chloroform suspensions of egg phosphatidylcholine, hydration of the lipid film with aqueous phases containing or not Nanotex, Rhodamine-100 dye or ω-3 PUFA-EE, and eleven extrusion steps through nanometric membrane filters. This resulted in uniform preparations of liposomes of approximately 200 nm diameter. Extraliposomal contents were removed from the preparation by gel filtration chromatography. High Resolution Magic Angle Spinning 1H NMR Spectroscopy of the liposomal preparations containing ω-3 PUFA-EE revealed well resolved 1H resonances from highly mobile ω-3 PUFA-EE, suggesting the formation of very small (ca. 10 nm) ω-3 PUFA-EE nanogoticules, tumbling fast in the NMR timescale. Chloroform extraction of the liposomal preparations revealed additionally the incorporation of ω-3 PUFA-EE within the membrane domain. Water diffusion weighted spectra, indicated that the goticules of ω-3 PUFA-EE or its insertion in the membrane did not affect the average translational diffusion coefficient of water, suggesting an intraliposomal localization, that was confirmed by ultrafiltration. The therapeutic efficacy of these preparations was tested in two different models of inflammatory disease as inflammatory colitis or the inflammatory component associated to glioma development. Results indicate that the magnetoliposomes loaded with ω-3 PUFA-EE allowed MRI visualization in vivo and improved the outcome of inflammatory disease in both animal models, decreasing significantly colonic inflammation and delaying, or even reversing, glioma development. Together, our results indicate that magnetoliposomes loaded with ω-3 PUFA-EE may become useful anti-inflammatory agents for image guided drug delivery.

Single-walled carbon nanotubes as anisotropic relaxation probes for magnetic resonance imaging†

We report on the preparation and characterization of magnetically oriented single-walled carbon nanotube arrangements as novel nanoprobes to enhance anisotropically water relaxation as detected by magnetic resonance imaging methods. SWCNT suspensions immobilized in agarose gels showed evident magnetic anisotropy with significantly longer T2 in the parallel than in the perpendicular orientations.

Amide conjugates of the DO3A-N-(α-amino)propionate ligand: leads for stable, high relaxivity contrast agents for MRI?

A novel synthetic methodology for preparing amide conjugates of the DO3A-N-(α-amino)propionate chelator is described, using the synthesis of the DO3A-N-(α-benzoylamido)propionate chelator as an illustrative example. The model Gd[DO3A-N-(α-benzoylamido)propionate] chelate displays accelerated water exchange, stability in a wide pH range and inertness towards transmetallation by Zn(2+). The Gd[DO3A-N-(α-benzoylamido)propionate] complex is mainly excreted via the kidneys, producing a significant increase in the kidney medulla/cortex enhancement ratio in MR images of Wistar rats, reflecting probably its higher lipophilicity compared with Gd(DTPA). The results presented suggest that Gd[DO3A-N-(α-amido)propionate] chelates can be valuable leads for preparing potentially safe high relaxivity MRI contrast agents.

Diffusion-Weighted Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is a technique based on the contents and relaxation features of water in tissues. In basic MRI sequences, diffusion phenomenon of water molecules is not taken into account although it has a notable influence in the relaxation times, and therefore in the signal intensity of images. In fact, MRI techniques that take advantage of water diffusion have experienced a huge development in last years. Diffusion-weighted imaging (DWI) has spectacularly evolved reaching nowadays a great impact both in clinical and preclinical imaging-especially in the neuroimaging field-and in basic research. We present here a protocol to perform DWI studies in a high-field preclinical setup.

Basic Pulse Sequences in Magnetic Resonance Imaging

Magnetic resonance images are obtained by a combination of different radiofrequency pulses and gradient waveforms applied to the subject inside a magnetic field. There are multiple pulse sequences used in clinical and preclinical studies adjusted to whatever physician or researches want to analyze, from basic anatomic images to accurate diagnostic techniques as diffusion, perfusion, or functional imaging. In this chapter, we present the most used radiofrequency pulse combinations of the two groups of sequences available in magnetic resonance imaging: spin-echo and gradient-echo sequences.

Novel contrast agents for multimodal biomedical imaging based in nanotechnology

Clinical imaging modalities have reached a prominent role in medical diagnosis and patient management in the last decades. Different image methodologies as Positron Emission Tomography, Single Photon Emission Tomography, X-Rays, or Magnetic Resonance Imaging are in continuous evolution to satisfy the increasing demands of current medical diagnosis. Progress in these methodologies has been favored by the parallel development of increasingly more powerful contrast agents. These are molecules that enhance the intrinsic contrast of the images in the tissues where they accumulate, revealing noninvasively the presence of characteristic molecular targets or differential physiopathological microenvironments. The contrast agent field is currently moving to improve the performance of these molecules by incorporating the advantages that modern nanotechnology offers. These include, mainly, the possibilities to combine imaging and therapeutic capabilities over the same theranostic platform or improve the targeting efficiency in vivo by molecular engineering of the nanostructures. In this review, we provide an introduction to multimodal imaging methods in biomedicine, the sub-nanometric imaging agents previously used and the development of advanced multimodal and theranostic imaging agents based in nanotechnology. We conclude providing some illustrative examples from our own laboratories, including recent progress in theranostic formulations of magnetoliposomes containing ω-3 poly-unsaturated fatty acids to treat inflammatory diseases, or the use of stealth liposomes engineered with a pH-sensitive nanovalve to release their cargo specifically in the acidic extracellular pH microenvironment of tumors.