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WIREs Nanomed Nanobiotechnol
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Methodologies and approaches for the analysis of cell–nanoparticle interactions

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How to study nanoparticle–cell interactions is the key question that puzzles researchers in the fields of nanomedicine as well as in nanotoxicology. In nanotoxicology, the amount of nanoparticles internalized by the cells or bound to the external surfaces of cells determines the toxic profile of those particles. In medical applications, cellular uptake and binding of medically effective nanoparticles decides their efficacy. Despite the importance of understanding the extent and mode of nanoparticle–cell interactions, these processes are underinvestigated, mainly due to the lack of suitable user‐friendly methodologies. Here we discuss the advantages and limitations of currently available (and most advanced) microscopic, spectroscopic, and other bioanalytical methods that could be used to assess cell‐nanoparticle interactions either qualitatively or quantitatively. Special emphasis is given to the methods that enable analysis and identification of nanoparticles at single‐cell level, and allow intracellular localization and speciation analysis of nanoparticles. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
Example imaging of nanoparticles and their cellular localization using fluorescence microscopy. Green fluorescent Ag nanoparticles exposed to human HepG2 cells were stained with DAPI (blue nuclear dye) and cell mask (red, membranes). Nanoparticles in cells are shown with white arrows. (Reprinted with permission from Ref . Copyright 2013 Creative Commons Attribution License (CC BY))
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Removal of cell surface bound Ag nanoparticles by hexacyanoferrate–thiosulphate redox‐based etchant. (a) Schematic of Ag nanoparticle removal from cell surface; (b) fluorescence microscopy visualization of Ag nanoparticle removal from cell surface. Ag nanoparticles were labeled with red fluorophore. In pre‐etching conditions, clear binding of Ag NPs to cell surface can be seen while after etching, only intracellular vesicular structures carrying fluorescent Ag nanoparticles can be observed. (Reprinted with permission from Ref . Copyright 2014 Nature Publishing Group)
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Localization of superparamagnetic iron oxide nanoparticles in the endosomes of cells. (a) Vesicles around nucleus (Nu), 4300× magnification; (b) detailed view of endosomes near nucleus, 8000× magnification. White arrow indicates the localization of endosomal nanoparticles. Scale bar: 1 µm. (Reprinted with permission from Ref . Copyright 2014 Elsevier Ltd.)
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Workflow of in vivo nanoparticle/cell interaction analysis using mass cytometry. (a) Nanoparticles are administered to experimental animal, (b) tissue from animal is isolated, (c) single cell suspension is prepared from tissue and stained with metal‐labeled probes, including antibodies, (d) single cell suspension is analyzed by mass cytometry, (e) output of mass cytometer is converted to Flow Cytometry Standard file, which allows either (f) manual or (g) machine‐learning based analysis of nanoparticle uptake by individual cells.
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A general scheme for quantitation of cell‐associated metal (Me) nanoparticles by mass spectrometry.
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Analysis of cell‐associated nonfluorescent nanoparticles by flow cytometry. (a) ARPE‐19 human retinal pigment epithelial cells were exposed to 30–40 nm TiO2 nanoparticles at different concentrations (3–30 µg/mL as indicated) and analyzed with flow cytometry. Cell‐associated nanoparticles can be inferred from increased side scatter signal (SSC) whereas cell size is measured with forward scatter (FSC) function. (Reprinted with permission from Ref . Copyright 2010 Wiley‐Liss, Inc.) (b) Correlation between flow cytometry SSC signal and mass of cell‐associated Ag (from ICP‐MS) in Ag nanoparticle‐exposed cells. Human T‐lymphocyte cells were exposed to Ag nanoparticles with different coatings: branched polyethylene imine (bPEI), citrate and polyethylene glycol (PEG) and different (10–70 nm) primary size. SSC signal of cells that were not exposed to Ag nanoparticles is shown for comparison. Dotted vertical line represents SSC value above which the signal was considered different from nonexposed cells. For 10 nm Ag NP exposed cells, only those which Ag content was above 100 fg (10,000 10 nm particles per cell) were SSC positive; for 30 nm Ag NP exposed cells those which Ag content was above 80 fg Ag (200 30 nm NPs per cell) were SSC positive; for 70 nm Ag NP exposed cells those which Ag content was above 20 fg (5 NPs per cell) were SSC positive. Figure is based on data presented in Ivask et al. (c) SSC signal of cells with 70 nm Ag particles attached to cell surface is shown on left image and SSC signal of cells with only intracellular nanoparticles is shown on right image. Exposure concentrations of Ag nanoparticles are indicated. (Reprinted with permission from Ref . Copyright 2016 Elsevier B.V.)
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Three‐dimensional reconstruction of 2D transmission electron microscope images. Visualization of intracellular quantum dots in human U‐2 OS osteosarcoma cell line. (Reprinted with permission from Refs and . Copyright 2013 American Chemical Society; 2014 Microscopy Society of America)
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Simultaneous fluorescence and dark‐field confocal scanning of TiO2nanoparticle‐exposed human bronchial epithelial BEAS 2B cells. Side view of cells exposed to TiO2 nanoparticles for 5 min (a) or 2 h (b). Scale bar 10 µm and magnification 3000×. Nucleus is stained with blue fluorescent dye and nanoparticles appear as bright spots. Red circles indicated the area from which slices in z‐axis (shown in (c) and (d)) were created. Lower graphs illustrate positioning of nanoparticles and nucleus in z axis in (a) (c) and (b) (d). (Reprinted with permission from Ref . Copyright 2011 BioMed Central Ltd.)
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Improvement in resolution using super‐resolution microscopes. (a) Outer membrane of mitochondria fluorescently immunolabeled against protein TOM20 imaged in confocal fluorescence microscope (up) and in stimulated emission depletion (STED) microscope (down) (Reprinted with permission from Ref . Copyright 2009 American Chemical Society). (b) Cell nucleus stained with DAPI (blue nuclear dye), Lamin B (green nuclear envelope marker), and for nuclear pore complex (red), visualized in confocal fluorescence microscope (up) and in structured illumination microscopy (SIM) mode. Left images represent the insets shown on right side images with (upper) or without (lower) Lamin B signal. (Reprinted with permission from Ref . Copyright 2008 American Association for the Advancement of Science)
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Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
Nanotechnology Approaches to Biology > Cells at the Nanoscale

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