Analytische Chemie
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Well-absorbed iron-based nanoparticulated materials are a promise for the oral management of iron deficient anemia. In this work, a battery of in vitro and in situ experiments are combined for the evaluation of the uptake, distribution and toxicity of new synthesized ultrasmall (4 nm core) Fe2O3 nanoparticles coated with tartaric/adipic acid with potential to be used as oral Fe supplements. First, the in vitro simulated gastric acid solubility studies by TEM and HPLC-ICP-MS reveal a partial reduction of the core size of about 40% after 90 min at pH3. Such scenario confirms the arrival of the nanoparticulate material in the small intestine. In the next step, the in vivo absorption through the small intestine by intestinal perfusion experiments is conducted using the sought nanoparticles in Wistar rats. The quantification of Fe in the NPs Suspension before and after perfusion shows Fe absorption levels above 79%, never reported for other Fe treatments. Such high absorption levels do not seem to compromise cell viability, evaluated in enterocytes-like models (Caco-2 and HT-29) using cytotoxicity, ROS production, genotoxicity and lipid peroxidation tests. Moreover, regional differences in terms of Fe concentration are obtained among different parts of the small intestine as duodenum>jejunum>ileum. Complementary transmission electron microscopy (TEM) images show the presence of the intact particles around the intestinal microvilli without significant tissue damage. These studies show the high potential of these NP preparations for their use as oral management of anemia.
Mycotoxins occur widely in foodstuffs and cause a variety of mold-related health risks to humans and animals. Elucidation of the metabolic fate of mycotoxins and the growing number of newly discovered mycotoxins have enhanced the demand for fast and reliable simulation methods. The viability of electrochemistry coupled with mass spectrometry (EC/ESI-MS), Fenton-like oxidation, and UV irradiation for the simulation of oxidative phase I metabolism of the mycotoxins citrinin (CIT) and dihydroergocristine (DHEC) was investigated. The specific reaction products are compared with metabolites produced by human and rat liver microsomes in vitro. Depending on the applied potential between 0 and 2000 mV vs. Pd/H-2 by using a flow-through cell, CIT and DHEC are oxidized to various products. Besides dehydrogenation and dealkylation reactions, several hydroxylated DHEC and CIT species are produced by EC and Fenton-like reaction, separated and analyzed by LC-MS/MS and ESI-HRMS. Compared to reaction products from performed microsomal incubations, several mono- and dihydroxylated DHEC species were found to be similar to the reaction products of EC, Fenton-like reaction, and UV-induced oxidation. Consequentially, nonmicrosomal efficient and economic simulation techniques can be useful in early-stage metabolic studies, even if one-to-one simulation is not always feasible.
An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos CPF)based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in-vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in-vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and Matrix complexity compared to conventional in-vivo or in-vitro methods.
SETNanoMetro, a European Seventh Framework project, seeks to develop standard synthetic routes and metrological characterisation methods for the development and production of TiO2 nanoparticles and nano-sized coatings with highly-defined, homogeneous and reproducible characteristics. These materials are being tested for their potential in selected technological applications, including as biomaterials, specifically as coatings on dental or orthopaedic metallic prostheses. This study aimed to assess how variations in nano-scale morphology and phase composition of TiO2 coatings affect their biocompatibility in vitro.
Pulsed DC magnetron sputtering was used to deposit a layer of Ti metal followed by a layer of TiO¬2 on standard glass microscope slides. The substrate bias voltage was varied during deposition to control the morphology and phase composition of the TiO2 layers. In order of increasing substrate bias voltage, the phase compositions of the TiO2 layers were: predominantly anatase, mixed anatase/rutile, and predominantly rutile, as confirmed by XRD. Examination of the coating cross-sections by SEM revealed feather-like columnar structures in the thin (950 nm thick) and thick (1550 nm thick) anatase coatings and in the mixed anatase/rutile coating (900 nm thick). In the rutile coating (730 nm thick), the columns were denser and had largely lost their feather-like structure. Top-view SEM showed square-pyramidal morphology of the columns in the anatase coatings, with columns generally 100 nm or smaller in size (thin coating) or up to 200 nm across (thick coating). In the mixed anatase/rutile coating, the top-view showed less regular columnar morphology with more elongated columns (up to approx. 100 nm by 200 nm). In the rutile coating, the top-view showed a less ordered, pebble-like morphology.
MG-63 human osteoblast-like cells and RAW 264.7 murine macrophage cells were cultured on the TiO2-coated substrates for 24 or 72 h before quantification of cell proliferation using the WST-1 cell proliferation assay. A toxic response was defined as a reduction in cell viability of greater than 30%. After 24 h culture, proliferation of MG-63 cells was significantly greater than the control (p < 0.05) on the thin anatase and mixed anatase/rutile coatings. After 72 h of culture no significant difference to the control was observed. For RAW 264.7 cells, proliferation was non-significantly decreased compared to the control on all coatings except the rutile coating after 24 h. After 72 h, RAW 264.7 proliferation was significantly decreased (p < 0.01) to 68% and 61% of the control for the thick and thin anatase coatings, respectively, indicating a toxic response. The results indicate that nano-sized TiO2 coatings show different biocompatibility to different test cell types, with a dependence upon coating phase composition and morphology.