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Graphene oxide (GO) materials possess physicochemical properties that facilitate their application in the industrial and medical sectors. The use of graphene may pose a threat to biota, especially aquatic life. In addition, the properties of nanomaterials can differentially affect cell and molecular responses. Therefore, it is essential to study and define the possible genotoxicity of GO materials to aquatic organisms and their ecosystems. In this study, we investigated the changes in the expression of 11 genes in the aquatic organism Chironomus riparius after 96 h of exposure to small GOs (sGO), large GOs (lGO) and monolayer GOs (mlGO) at 50, 500 and 3000 μg/L. Results showed that the different genes encoding heat shock proteins (hsp90, hsp70 and hsp27) were overexpressed after exposure to these nanomaterials. In addition, ATM and NLK—the genes involved in DNA repair mechanisms—were altered at the transcriptional level. DECAY, an apoptotic caspase, was only activated by larger size GO materials, mlGO and lGO. Finally, the gene encoding manganese superoxide dismutase (MnSOD) showed higher expression in the mlG O-treated larvae. The lGO and mlGO treatments indicated high mRNA levels of a developmental gene (FKBP39) and an endocrine pathway-related gene (DRONC). These two genes were only activated by the larger GO materials. The results indicate that larger and thicker GO nanomaterials alter the transcription of genes involved in cellular stress, oxidative stress, DNA damage, apoptosis, endocrine and development in C. riparius. This shows that various cellular processes are modified and affected, providing some of the first evidence for the action mechanisms of GOs in invertebrates. In short, the alterations produced by graphene materials should be further studied to evaluate their effect on the biota to show a more realistic scenario of what is happening at the molecular level.

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.

Graphene oxide (GO) is a carbon nanomaterial used in electronics, biomedicine, environmental remediation and biotechnology. The production of graphene will increase in the upcoming years. The carbon nanoparticles (NPs) are released into the environment and accumulated in aquatic ecosystems. Information on the effects of GO in aquatic environments and its impact on organisms is still lacking. The aim of this study was to synthesise and characterise label-free GO with controlled lateral dimensions and thickness - small GO (sGO), large GO (lGO) and monolayer GO (mlGO) - and determine their impact on Chironomus riparius, a sentinel species in the freshwater ecosystem. Superoxide dismutase (SOD) and lipid peroxidation (LPO) was evaluated after exposures for 24 h and 96 h to 50, 500, and 3000 μg/L. GOs accumulated in the gut of C. riparius and disturbed its antioxidant metabolism. We suggest that all types of GO exposure can upregulate of SOD. Moreover, both lGO and mlGO treatments caused LPO damage in C. riparius in comparison to sGO, proving its favourable lateral size impact in this organism. Our results indicate that GOs could accumulate and induce significant oxidative stress on C. riparius. This work shows new information about the potential oxidative stress of these NMs in aquatic organisms.

We report on the magnetic properties of stable suspensions from oxidized Multiwalled Carbon Nanotubes (MWCNT) functionalized with aminopyrene (AP). MWCNT form π−π stacking adducts with AP (AP-MWCNT), originating homogenous, stable, suspensions in N,N-dimethylformamide (DMF) or melted agarose. First, we investigated the magneto-optical properties of these adducts. When applying series of pulsed magnetic fields to nanotube suspensions in DMF, the pattern of light dispersed increased during the magnetic pulse and decreased in the intervals, a behavior consistent with magnetic field induced orientation of the adducts. When adducts were suspended in a melted agarose gel under an external magnetic field, the extinction coefficient of polarized light through the gel, was larger when the polarization plane was parallel to the magnetic field direction. Based on the magneto-optical responses observed, we further investigated the magnetic properties of AP-MWCNT implementing measurements with Superconducting Quantum Interference Device, Zero Field Cooling and Field Cooling, Thermogravimetric and Differential Scanning Calorimetry. Pre-oriented AP-MWCNT suspensions depicted a clear superparamagnetic character with hysteresis loops revealing larger magnetic susceptibility values along their longitudinal axis. In summary, magneto-optical and SQUID measurements revealed that nanotube adducts in suspension, behave as nanoscale compass needles aligning their long axis parallel to externally applied magnetic fields., This work was supported in part by grants SAF2014-53739-R/IPT-2012-06000/SAF2017-08343-R (to SC), CTQ-2013-47669-R (to PB), MAT2013-43301-R and MAT2016-76106-R (to M O Ramírez) from the Spanish Ministry of Economy and Competitiveness and grants S2010/BMD-2349 and B2017/BMD-3688 (to PB and SC) and S2013/MIT-2740 (to M O Ramírez) from the Community of Madrid. DC and VN held postdoctoral contracts from CSIC. CM acknowledges the financial support by the “Ramón y Cajal” Program of MINECO RYC-2014-16626.

This work are distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License., 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., This work was supported in part by grants from SOLUTEX SL to SC, grants from the Spanish Ministry of Economy and Competitivity SAF2011-23622, IPT-2012-1331-006000 to SC, grant CTQ2013-47669-R to PB, and grant S2010/BMD-2349 from the Community of Madrid to S.C. and P.B. D.C. and V.N. held predoctoral contracts from Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC).

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 C-13 hiperpolarization 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 mutimodal and molecular imaging approaches are discussed., This work was supported in part by grants: CTQ2006-06505/BQU, CTQ2009-14146-C01/C02, to P.B. and P.L.L., S-BIO/0179/2006 and SAF2008/01381 to SC and EU MEDITRANS Integrated Project NMP4-CT- 2006-02668 to S.C., P.L.L. and P.B. J. P- T, D. C., B.L. and V.N. are predoctoral fellows from the Community of Madrid and CSIC and V.N. and C.U hold contracts from UNED

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.

25 pages, 7 figures, 25 schemes., 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., This work was supported in part by: CTQ2006-06505/BQU to PB, SAF 2004-02145, NAN2004- 09125-C07-03, FIS C03/10, FIS C03/08, FIS G03/155 to SC, European Community MEDITRANS 2006; Integrated EU Project FP6-2004-NMP-NI-4/IP 026668-2 and S-BIO/0179/2006 from the Community of Madrid to PB and SC.

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 Fick's law, while water diffusion inside the nanotubes is not described by a Fick's 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., We thank grants CTQ2013-47699-R, 2013/UEM11 and 2014/UEM14.

Exogenous nanotubular materials can be used to induce and perturb selectively the anisotropic diffusion of water molecules in the surrounding medium in a manner detectable by magnetic resonance imaging (see scheme; ADC: apparent translational diffusion coefficient). Nanotubes may be suitable contrast agents in studies of the microstructural basis of tissue anisotropy and its pathological implications., This work was supported in part by grants CTQ2006-06505/BQU, CTQ2009-14146-C01/C02, NAN2004-09125-C07-03, S-BIO/0179/2006, and SAF2008/01381, and by the EU (MEDITRANS Integrated Project NMP4-CT-2006-02668 to S.C., P.L.L., and P.B.). V.N. holds a JAE predoctoral fellowship from CSIC.

To design efficient targeting strategies in magnetic resonance (MR) molecular imaging applications, the formation of supramolecular adducts between (strept)avidin ((S)Av) and tribiotinylated Gd-DOTA-monoamide complexes (DOTA=1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was explored. Two compounds based on the trivalent core of tris(2-aminoethyl)amine each containing three biotin molecules and one (L1) or three (L2) DOTA-monoamide (DOTAMA) ligands were synthesized. In these tribiotinylated derivatives the biotins are spaced far enough apart to allow the formation of the supramolecular adduct with the protein and to host the chelating units in between the (S)Av layers. Size exclusion HPLC analyses indicated complete formation of very high molecular weight polymers (>2 MDa) with (S)Av in solution. A (1)H NMR spectroscopy relaxometric study on the obtained polymeric adducts showed a marked increase of the relaxivity at 35-40 MHz as a consequence of the lengthening of the tumbling time due to the formation of Gd-chelates/(S)Av polymers. The most efficient Gd(3)L2/(S)Av polymeric system was used for a test in cell cultures. The target is represented by a neural cell adhesion molecule (NCAM), which is overexpressed in Kaposi's sarcoma cells and tumor endothelial cells (TEC) and that is efficiently recognized by a biotinylated tetrameric peptide (C3d-Bio). In vitro experiments showed that only cells incubated with both C3d-Bio and Gd(3)L2/SAv polymer were hyperintense with respect to the control. Relaxation rates of cell pellets incubated with Gd(3)L2/SAv alone were not significantly different from the untreated cells demonstrating the absence of a specific binding.

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.

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.