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WIREs Nanomed Nanobiotechnol
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High throughput toxicity screening and intracellular detection of nanomaterials

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With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
(a) Experimental workflow and (b) experimental design for effective high‐throughput screening
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Savings in time and cost with HTS comet assay.
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Tungsten carbide‐cobalt (WC‐Co) NP genotoxicity determined by measuring foci of γ‐H2Ax (phosphorylated H2Ax histones), which are directly proportional to the number of DNA double‐strand breaks. Counts of γ‐H2Ax foci were performed on at least 200 cells per condition and the results are depicted as box plot distribution values [minimum (min), maximum (max), median, 25th and 75th percentiles] of the number of foci obtained for each tested condition. A Wilcoxon rank test was performed for statistical comparisons (i.e., vs. control cells not exposed to NPs; *p < 0.01). For both cell lines Caki‐1 and Hep3B, WC‐Co NPs were found to be genotoxic in a dose‐dependent manner (b). For γ‐H2Ax positive control, Caki‐1 cells were exposed to γ irradiation (a).
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Examples of high throughput equipment. (a) Apparatus for performing comet assay on 12 minigels on one slide. Right: Component parts of 12‐gel chamber unit (Severn Biotech, Kidderminster, UK), including metal base with marks for positioning gels on slide, silicone rubber gasket, plastic top‐plate with wells, and silicone rubber seal. Left: chamber unit assembled. (b) The xCELLigence® instruments (ACEA Biosciences Inc, San Diego, CA, USA) employ impedance‐based label‐free real‐time monitoring of cells. Cell number, proliferation, viability, morphology, and adhesion are quantified. Electronic microtiter plates: 16‐well (left), 96‐well (middle), and 384‐well (not shown), can be used for high throughput nanotoxicity screening. The instrument is placed in a standard CO2 incubator and is cable‐connected with analysis and control units outside the incubator (right). The data and the performance of the instrument are displayed real‐time (right). (c) The Ampha™ Z30 impedance flow cytometer (left) (Amphasys AG, Lucerne, Switzerland) uses microfluidic chips with microelectrodes (right) to measure changes in the electrical resistance of the fluid, in which cells are suspended, when cells pass through the applied AC electric field. Cell viability and mode of cell death (apoptosis vs. necrosis) can be detected and quantified. (d) GE Healthcare Cytell Cell Imaging System captures cellular and sub‐cellular images in a benchtop unit equipped with on‐board data analysis and visualization tools. It streamlines and simplifies routine assays, such as cell cycle and cell viability assays, to save time and help research progress more rapidly.
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Impedance‐based measurements of U937 monoblastoid cells exposed to NM 300 K silver particles (15 nm, spherical): Representative data collected with the Ampha Z30 microchip‐based flow cytometer (Amphasys AG, Switzerland) (See Figure for illustration). The figure shows the dotplots of (a) necrotic cells (heated at 70°C), (b) unexposed cells, and (c) cells exposed for 24 h to 100 µg/mL NM 300 k.
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Impedance‐based measurements: cell index (CI) real‐time monitoring and viability of cells exposed to WC‐Co NPs. Index real‐time monitoring and viability of A549 (a), Caki‐1 (b), and Hep3B (c) cells exposed to tungsten carbide‐cobalt (WC‐Co) NPs. Impedance measurements (one representative experiment among three independent experiments) were carried out for 72 h and cell indices were normalized at time 0 to ensure no inter‐well variability prior to the addition of NPs. Control cells were not exposed to WC‐Co NPs. Positive control cells were exposed for 72 h to 0.005% Triton in the case of Caki‐1 and Hep3B cells and to 0.01% Triton for A549 cells. The histograms correspond to CI values at three endpoints (24, 48, and 72 h) for control cells, to a positive control (Triton), and to cells exposed to 1, 5, 25, 50, 75, 100, and 150 µg/mL of WC‐Co NPs. Statistical analysis was performed for each exposure condition compared to nonexposed cells (Student's t‐test, *p < 0.01; **p < 0.001; ***p < 0.0001).
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Impedance‐based measurements of A549 cells exposed to NM‐100 titanium dioxide particles (110 nm diameter, anatase): Representative data collected with the xCELLigence instrument (ACEA Biosciences, USA) (See Figure for illustration). The figure shows the plot of the cell index (CI) which reflects real‐time cellular proliferation. Exposure of cells started at 24 h. The conditions are color coded from green to red in concentrations 0, 2, 10, 20, 50, and 100 µg/mL. Medium only (black) or with 100 µg/mL NM‐100 (blue) are included for reference.
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Forward scatter (FS)/side scatter (SS) plots to determine NP uptake in 3T3 cell line after 24 h CeO2 NP exposure. Blue box, cells with no NP incorporation; Red box, uptake of NPs by 3T3 cells. (a) Control group with no CeO2 NPs. (b) CeO2 NP exposure at 0.01 mg/mL. (c) CeO2 NP exposure at 0.1 mg/mL.
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Flow cytometric detection of reactive oxygen species produced in 3T3 cells after 24 h CeO2 NP exposure. FL1 represents fluorescence from oxidation of chloromethyldichlorodihydrofluorescein diacetate (CM‐H2DCFDA). Blue area, control group without CeO2 NP exposure; Red area, 0.1 mg/mL CeO2 NP exposure.
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Micro‐proton‐induced X‐ray emission (μPIXE) elemental mapping of A549 cells exposed to different metal‐oxide NMs at a concentration of 30 µg/mL for 48 h. Top and bottom images demonstrate S (Sulfur) and NM related element distributions, respectively. The color code is as follows: yellow is the maximum, black represents the minimum. The size of all images is 50 × 50 µm. (Reprinted with permission from Ref . Copyright 2014 Wiley)
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Therapeutic Approaches and Drug Discovery > Emerging Technologies
Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine
Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

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