title: Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy.
abstract: Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes.
authors.affiliation: a Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration , Silver Spring , MD , USA .
authors.forename: Bridget E
authors.initials: BE
authors.lastname: Wildt
authors.affiliation: b Pontificia Universidad Catolica De Chile , Santiago , Chile .|c Scanogen Inc , Baltimore , MD , USA , and.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Celedon
authors.affiliation: d Applied Biotechnology Branch, 711th Human Performance Wing, Wright-Patterson AFB , Dayton , OH , USA.
authors.forename: Elizabeth I
authors.initials: EI
authors.lastname: Maurer
authors.affiliation: a Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration , Silver Spring , MD , USA .
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: Casey
authors.affiliation: a Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration , Silver Spring , MD , USA .
authors.forename: Amber M
authors.initials: AM
authors.lastname: Nagy
authors.affiliation: d Applied Biotechnology Branch, 711th Human Performance Wing, Wright-Patterson AFB , Dayton , OH , USA.
authors.forename: Saber M
authors.initials: SM
authors.lastname: Hussain
authors.affiliation: a Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration , Silver Spring , MD , USA .
authors.forename: Peter L
authors.initials: PL
authors.lastname: Goering
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D053768:Metal Nanoparticles / Q000633:toxicity*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010316:Particle Size; D012834:Silver / Q000378:metabolism / Q000633:toxicity*; D012995:Solubility; D013499:Surface Properties
chemical_list: D012834:Silver; C018533:ethidium homodimer; D004996:Ethidium
keywords: Cellular uptake; ICP-MS; confocal microscopy; image analysis; nanosilver; silver ions; silver nanoparticles; transmission electron microscopy