Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), M(W) = 2000 g mol(-1)). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l(-1)). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence.Solid-state NMR spectroscopy is unique in providing information at atomic resolution of membrane polypeptides in a native bilayer environment. The technique has been used for investigations of their structure, topology, dynamics and heterogeneous nature in phospholipid bilayers [1][2][3][4][5][6][7][8][9] . However, NMR suffers from its inherently low signal intensity and the investigation of membrane-associated polypeptides turns out particularly difficult due to the dilution of peptide in lipid (typically around 1-2 mole%) and the increased line width due to inhomogeneous line broadening. Although these limitations are most pronounced when static samples are investigated the large anisotropy of NMR chemical shift, dipolar and quadrupolar interactions has been successively used to determine the structure and/or topology of a number of peptides in oriented lipid bilayers 1,2,8,9 . These could be refined by additional distance restraints for example from magic angle sample spinning (MAS) solid-state NMR spectra 1,2,8,9 . During the last years considerable progress has been made in dynamic nuclear polarization (DNP) solid-state NMR and the technique has been shown to overcome many of the sensitivity limitations of solid-state NMR spectroscopy [10][11][12][13] . During DNP/solid-state NMR experiments the radicals dispersed in the sample result in a high electron polarization which is transferred to the 1 H bath through microwave irradiation. In a next step cross-polarization to the heteronuclei assures enhancements of the NMR signal typically by about two orders of magnitude 10,14 . Also in the case of membrane samples 4,11,15,16 or of membrane proteins in cellular environments considerable improvements have been obtained 17 .

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h.

International audienceDynamic nuclear polarization (DNP)/solid-state nuclear magnetic resonance(NMR) spectroscopy bears great potential for the investigation of membraneassociatedpolypeptides which can often be produced only in small amounts and whichneed to be ‘diluted’ in lipid bilayer environments to adopt or maintain their functionalstructure. Here we present investigations using biradicals, such as TOTAPOL andbTbK, for solid-state NMR signal enhancement using DNP in the context of lipidmembranes. By transferring polarization from electron to nuclear spins using microwave irradiation signal enhancement factors of up to 13 are obtained withTOTAPOL and up to 17 with bTbK. The possible reasons why these factors are belowthose obtained in glassy samples of bulk solvents (40–60 under similar conditions) areevaluated and discussed. In order to further ameliorate the enhancement factors thephysico-chemical characteristics of TEMPOL, TOTAPOL, bTbK, and bCTbK, suchas their partitioning between hydrophilic and hydrophobic solvents or their stabilityunder different environmental conditions are presented. Finally, having providedproof-of-concept that DNP/solid-state NMR measurements can be performed withoriented membrane samples work in progress is presented on the development of a flatcoilprobe for DNP/solid-state NMR experiments on oriented membranes

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