Effects at molecular level of multi-walled carbon nanotubes (MWCNT) in Chironomus riparius (DIPTERA) aquatic larvae.

Nowadays, due to the physical, chemical, electrical, thermal and mechanical properties of carbon nanotubes (CNT), its have been currently incorporated into biomedical products and they are employed in drug delivery drug administration, biosensor design, microbial treatments, consumer products, and new products containing CNT are expected in the future. CNT are hydrophobic and have a tendency to accumulate in sediments if they are released into aquatic ecosystems. Vertebrate studies have revealed concerns about the toxicity of carbon nanotubes, but there is very limited data on the toxic effects in aquatic invertebrate species. The aim of the present study is to determine the effects of MWCNT in Chironomus riparius at the molecular level, understanding its mode of action and analyzing the suitability of this species to monitor and assess risk of nanomaterials in aquatic ecosystems. To evaluate possible toxic effects caused by carbon nanotube environmental dispersion with regard to aquatic compartment, we study the mRNA levels of several related genes with DNA repairing mechanisms, cell stress response, cell apoptosis and cytoskeleton by Real-Time PCR and proposed a freshwater invertebrate C. riparius, which is a reference organism in aquatic toxicology. The obtained results show a transcriptional alteration of some genes included in this study, indicating that different cell processes are affected and providing one the first evidences in the mechanisms of action of MWCNT in invertebrates. Moreover, this data reinforces the need for further studies to assess the environmental risk of nanomaterial to prevent future damage to aquatic ecosystems.

Advanced Contrast Agents for Multimodal Biomedical Imaging Based on 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.

Coarse-grained molecular dynamics simulation of water diffusion in the presence of carbon nanotubes.

Computational modeling of the translational diffusion of water molecules in anisotropic environments entails vital relevance to understand correctly the information contained in the magnetic resonance images weighted in diffusion (DWI) and of the diffusion tensor images (DTI). In the present work we investigated the validity, strengths and weaknesses of a coarse-grained (CG) model based on the MARTINI force field to simulate water diffusion in a medium containing carbon nanotubes (CNTs) as models of anisotropic water diffusion behavior. We show that water diffusion outside the nanotubes follows Ficks law, while water diffusion inside the nanotubes is not described by a Ficks behavior. We report on the influence on water diffusion of various parameters such as length and concentration of CNTs, comparing the CG results with those obtained from the more accurate classic force field calculation, like the all-atom approach. Calculated water diffusion coefficients decreased in the presence of nanotubes in a concentration dependent manner. We also observed smaller water diffusion coefficients for longer CNTs. Using the CG methodology we were able to demonstrate anisotropic diffusion of water inside the nanotube scaffold, but we could not prove anisotropy in the surrounding medium, suggesting that grouping several water molecules in a single diffusing unit may affect the diffusional anisotropy calculated. The methodologies investigated in this work represent a first step towards the study of more complex models, including anisotropic cohorts of CNTs or even neuronal axons, with reasonable savings in computation time.

Image guided drug release from pH-sensitive Ion channel-functionalized stealth liposomes into an in vivo glioblastoma model.

Liposomal drug delivery vehicles are promising nanomedicine tools for bringing cytotoxic drugs to cancerous tissues selectively. However, the triggered cargo release from liposomes in response to a target-specific stimulus has remained elusive. We report on functionalizing stealth-liposomes with an engineered ion channel and using these liposomes in vivo for releasing an imaging agent into a cerebral glioma rodent model. If the ambient pH drops below a threshold value, the channel generates temporary pores on the liposomes, thus allowing leakage of the intraluminal medicines. By using magnetic resonance spectroscopy and imaging, we show that engineered liposomes can detect the mildly acidic pH of the tumor microenvironment with 0.2 pH unit precision and they release their content into C6 glioma tumors selectively, in vivo. A drug delivery system with this level of sensitivity and selectivity to environmental stimuli may well serve as an optimal tool for environmentally-triggered and image-guided drug release. FROM THE CLINICAL EDITOR: Cancer remains a leading cause of mortality worldwide. With advances in science, delivery systems of anti-cancer drugs have also become sophisticated. In this article, the authors designed and characterized functionalized liposomal vehicles, which would release the drug payload in a highly sensitive manner in response to a change in pH environment in an animal glioma model. The novel data would enable better future designs of drug delivery systems.

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 (1)H NMR Spectroscopy of the liposomal preparations containing ω-3 PUFA-EE revealed well resolved (1)H 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.

Hypothalamic metabolic compartmentation during appetite regulation as revealed by magnetic resonance imaging and spectroscopy methods.

We review the role of neuroglial compartmentation and transcellular neurotransmitter cycling during hypothalamic appetite regulation as detected by Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) methods. We address first the neurochemical basis of neuroendocrine regulation in the hypothalamus and the orexigenic and anorexigenic feed-back loops that control appetite. Then we examine the main MRI and MRS strategies that have been used to investigate appetite regulation. Manganese-enhanced magnetic resonance imaging (MEMRI), Blood oxygenation level-dependent contrast (BOLD), and Diffusion-weighted magnetic resonance imaging (DWI) have revealed Mn(2+) accumulations, augmented oxygen consumptions, and astrocytic swelling in the hypothalamus under fasting conditions, respectively. High field (1)H magnetic resonance in vivo, showed increased hypothalamic myo-inositol concentrations as compared to other cerebral structures. (1)H and (13)C high resolution magic angle spinning (HRMAS) revealed increased neuroglial oxidative and glycolytic metabolism, as well as increased hypothalamic glutamatergic and GABAergic neurotransmissions under orexigenic stimulation. We propose here an integrative interpretation of all these findings suggesting that the neuroendocrine regulation of appetite is supported by important ionic and metabolic transcellular fluxes which begin at the tripartite orexigenic clefts and become extended spatially in the hypothalamus through astrocytic networks becoming eventually MRI and MRS detectable.

Dendritic MRI contrast agents: an efficient prelabeling approach based on CuAAC.

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) allows the efficient and complete functionalization of dendrimers with preformed Gd chelates (prelabeling) to give monodisperse macromolecular contrast agents (CAs) for magnetic resonance imaging (MRI). This monodispersity contrasts with the typical distribution of materials obtained by classical routes and facilitates the characterization and quality control demanded for clinical applications. The potential of a new family of PEG-dendritic CA based on a gallic acid-triethylene glycol (GATG) core functionalized with up to 27 Gd complexes has been explored in vitro and in vivo, showing contrast enhancements similar to those of Gadomer-17, which reveals them to be a promising platform for the development of CA for MRI.

Imaging tumor hypoxia by magnetic resonance methods.

Tumor hypoxia results from the negative balance between the oxygen demands of the tissue and the capacity of the neovasculature to deliver sufficient oxygen. The resulting oxygen deficit has important consequences with regard to the aggressiveness and malignancy of tumors, as well as their resistance to therapy, endowing the imaging of hypoxia with vital repercussions in tumor prognosis and therapy design. The molecular and cellular events underlying hypoxia are mediated mainly through hypoxia-inducible factor, a transcription factor with pleiotropic effects over a variety of cellular processes, including oncologic transformation, invasion and metastasis. However, few methodologies have been able to monitor noninvasively the oxygen tensions in vivo. MRI and MRS are often used for this purpose. Most MRI approaches are based on the effects of the local oxygen tension on: (i) the relaxation times of (19)F or (1)H indicators, such as perfluorocarbons or their (1)H analogs; (ii) the hemodynamics and magnetic susceptibility effects of oxy- and deoxyhemoglobin; and (iii) the effects of paramagnetic oxygen on the relaxation times of tissue water. (19)F MRS approaches monitor tumor hypoxia through the selective accumulation of reduced nitroimidazole derivatives in hypoxic zones, whereas electron spin resonance methods determine the oxygen level through its influence on the linewidths of appropriate paramagnetic probes in vivo. Finally, Overhauser-enhanced MRI combines the sensitivity of EPR methodology with the resolution of MRI, providing a window into the future use of hyperpolarized oxygen probes.

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.

Noninvasive multiparametric imaging of metastasis-permissive microenvironments in a human prostate cancer xenograft.

Metastasis continues to be one of the major causes of mortality from prostate cancer. Because human malignant cell lines metastasize more readily from orthotopic sites than from heterotopic sites, to identify metastasis-permissive tumor microenvironments, we used noninvasive imaging to compare the in vivo vascular, metabolic, and physiologic characteristics of a human prostate cancer xenograft implanted orthotopically in the prostate or s.c. in the flank. Hypoxia was detected in these xenografts by placing an enhanced green fluorescence protein optical reporter under the control of a hypoxia response element. A multiparametric analysis of hypoxia, extracellular pH, vascularization, and metabolism provided a characterization of environments that are permissive for metastasis to occur. We found that orthotopic tumors, which metastasized more easily, were characterized by higher vascular volume, permeability, and total choline and a more acidic extracellular pH. Interestingly, metastatic deposits in the lymph nodes as well as cancer cells in ascites fluid were found to be hypoxic, explaining, in part, the refractory nature of metastatic disease. These results also provide the basis for clinically translatable noninvasive imaging markers for predicting metastatic risk in prostate cancer.

The turnover of the H3 deuterons from (2-13C) glutamate and (2-13C) glutamine reveals subcellular trafficking in the brain of partially deuterated rats.

We investigated by 13C NMR the turnover of the H3 deuterons of (2-13C) glutamate and (2-13C) glutamine in the brain of partially deuterated rats. Adult animals (150-200 g) fed ad libitum received 50% 2H2O or tap water 9 days before infusing (1-13C) glucose or (2-13C) acetate for 5, 10, 15, 30, 60, or 90 min. The brains were then funnel-frozen and acid extracts were prepared and analyzed by high-resolution 13C NMR. The deuteration of one or the two H3 hydrogens of (2-13C) glutamate or glutamine resulted in single (-0.07 ppm) or double (-0.14 ppm) isotopic shifts upfield of the corresponding C2 perprotonated resonance, demonstrating two sequential deuteration steps. The faster monodeuteration generated 3R or 3S (2-13C, 3-2H) glutamate or glutamine through the alternate activities of cerebral aconitase or isocitrate dehydrogenase, respectively. The slower process produced bideuterated (2-13C, 3,3'-2H2) glutamate or glutamine through the consecutive activity of both enzymes. The kinetics of deuteration was fitted to a Michaelis-Menten model including the apparent K(m)' and Vmax' values for the observed deuterations. Our results revealed different kinetic constants for the alternate and consecutive deuterations, suggesting that these processes were caused by the different cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, respectively. The deuterations of (2-13C) glutamate or glutamine followed also different kinetics from (1-13C) glucose or (2-13C) acetate, revealing distinct deuteration environments in the neuronal or glial compartments.

Prótesis aórtica biológica de implantación percutánea: imágenes de necropsia / Percutaneous aortic bioprosthesis: necropsy images

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Chemistry of paramagnetic and diamagnetic contrast agents for Magnetic Resonance Imaging and Spectroscopy pH responsive contrast agents.

We provide a brief overview of the chemistry and most relevant properties of paramagnetic and diamagnetic contrast agents (CAs) for Magnetic Resonance Imaging and Magnetic Resonance Spectroscopic Imaging. Paramagnetic CAs for MRI consist mainly of Gd(III) complexes from linear or macrocyclic polyaminopolycarboxylates. These agents reduce, the relaxation times T(1) and T(2) of the water protons in a concentration dependent manner, increasing selectively MRI contrast in those regions in which they accumulate. In most instances they provide anatomical information on the localization of lesions and in some specific cases they may allow to estimate some physiological properties of tissues including mainly vascular performance. Because of its ability to discriminate easily between normal and diseased tissue, extracellular pH (pH(e)) has been added recently, to the battery of variables amenable to MRI investigation. A variety of Gd(III) containing macrocycles sensitive to pH, endogenous or exogenous polypeptides or even liposomes have been investigated for this purpose, using the pH dependence of their relaxivity or magnetization transfer rate constant (chemical exchange saturation transfer, CEST). Many environmental circumstances in addition to pH affect, however, relaxivity or magnetization transfer rate constants of these agents, making the results of pH measurements by MRI difficult to interpret. To overcome these limitations, our laboratory synthesized and developed a novel series of diamagnetic CAs for Magnetic Resonance Spectroscopic Imaging, a new family of monomeric and dimeric imidazolic derivatives able to provide unambiguous measurements of pH(e), independent of water relaxivity, diffusion or exchange.

Synthetic approaches to heterocyclic ligands for Gd-based MRI contrast agents.

Magnetic Resonance Imaging (MRI) methods are currently used in the clinic for the non invasive detection and characterization of a wide variety of pathologies. Increases in the diagnostic efficiency of MRI have been helped by both the design of dedicated MR sequences revealing specific aspects of the pathology and by the development of more sensitive and selective Contrast Agents (CAs), capable of more precisely delineating the borderline regions. In the present review we focus on the synthetic strategies used to obtain MRI CAs containing heterocyclic rings.

Serial in vivo spectroscopic nuclear magnetic resonance imaging of lactate and extracellular pH in rat gliomas shows redistribution of protons away from sites of glycolysis.

The acidity of the tumor microenvironment aids tumor growth, and mechanisms causing it are targets for potential therapies. We have imaged extracellular pH (pHe) in C6 cell gliomas in rat brain using 1H magnetic resonance spectroscopy in vivo. We used a new probe molecule, ISUCA [(+/-)2-(imidazol-1-yl)succinic acid], and fast imaging techniques, with spiral acquisition in k-space. We obtained a map of metabolites [136 ms echo time (TE)] and then infused ISUCA in a femoral vein (25 mmol/kg body weight over 110 min) and obtained two consecutive images of pHe within the tumor (40 ms TE, each acquisition taking 25 min). pHe (where ISUCA was present) ranged from 6.5 to 7.5 in voxels of 0.75 microL and did not change detectably when [ISUCA] increased. Infusion of glucose (0.2 mmol/kg.min) decreased tumor pHe by, on average, 0.150 (SE, 0.007; P < 0.0001, 524 voxels in four rats) and increased the mean area of measurable lactate peaks by 54.4 +/- 3.4% (P < 0.0001, 287 voxels). However, voxel-by-voxel analysis showed that, both before and during glucose infusion, the distributions of lactate and extracellular acidity were very different. In tumor voxels where both could be measured, the glucose-induced increase in lactate showed no spatial correlation with the decrease in pHe. We suggest that, although glycolysis is the main source of protons, distributed sites of proton influx and efflux cause pHe to be acidic at sites remote from lactate production.

Novel generation of pH indicators for proton magnetic resonance spectroscopic imaging.

We describe the synthesis of 1,omega-di-1H-imidazoles 2 and 3, derived from l-threitol and d-mannitol, respectively, showing suitable magnetic and toxicological properties, as novel extracellular pH indicators for 1H spectroscopic imaging by magnetic resonance methods.

Kinetic properties of the redox switch/redox coupling mechanism as determined in primary cultures of cortical neurons and astrocytes from rat brain.

We investigate the mechanisms underlying the redox switch/redox coupling hypothesis by characterizing the competitive consumption of glucose or lactate and the kinetics of pyruvate production in primary cultures of cortical neurons and astrocytes from rat brain. Glucose consumption was determined in neuronal cultures incubated in Krebs ringer bicarbonate buffer (KRB) containing 0.25-5 mM glucose, in the presence and absence of 5 mM lactate as an alternative substrate. Lactate consumption was measured in neuronal cultures incubated with 1-15 mM lactate, in the presence and absence of 1 mM glucose. In both cases, the alternative substrate increased the K(m) (mM) values for glucose consumption (from 2.2 +/- 0.2 to 3.6 +/- 0.1) or lactate consumption (from 7.8 +/- 0.1 to 8.5 +/- 0.1) without significant changes on the corresponding V(max). This is consistent with a competitive inhibition between the simultaneous consumption of glucose and lactate. When cultures of neurons or astrocytes were incubated with increasing lactate concentrations 1-20 mM, pyruvate production was observed with K(m) (mM) and V(max) (nmol/mg/h) values of 1.0 +/- 0.1 and 109 +/- 4 in neurons, or 0.28 +/- 0.1 and 342 +/- 54 in astrocytes. Thus, astrocytes or neurons are able to return to the incubation medium as pyruvate, a significant part of the lactate consumed. Present results support the reversible exchange of reducing equivalents between neurons and astrocytes in the form of lactate or pyruvate. Monocarboxylate exchange is envisioned to operate under near equilibrium, with the transcellular flux directed thermodynamically toward the more oxidized intracellular redox environment.

Experimental and theoretical study of lanthanide complexes based on linear and macrocyclic polyaminopolycarboxylic acids containing pyrazolylethyl arms.

We report the synthesis of two novel Gd(III)-complexes derived from linear and macrocyclic polyaminopolycarboxylic acids 1 and 2, which contain a 3,5- dimethylpyrazolyl-ethyl arm, and a study of their relaxivity properties. The relationships between the experimental and theoretical results have provided interesting information about the kinetic and thermodynamic stability of these complexes.