title: Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells.
abstract: Zinc oxide nanoparticles (ZnO NPs) exhibit novel physiochemical properties and have found increasing use in sunscreen products and cosmetics. The potential toxicity is of increasing concern due to their close association with human skin. A time-of-flight secondary ion mass spectrometry (TOF-SIMS) and confocal laser scanning microscopy (CLSM) imaging method was developed and validated for rapid and sensitive cytotoxicity study of ZnO NPs using human skin equivalent HaCaT cells as a model system. Assorted material, chemical, and toxicological analysis methods were used to confirm their shape, size, crystalline structure, and aggregation properties as well as dissolution behavior and effect on HaCaT cell viability in the presence of various concentrations of ZnO NPs in aqueous media. Comparative and correlative analyses of aforementioned results with TOF-SIMS and CLSM imaging results exhibit reasonable and acceptable outcome. A marked drop in survival rate was observed with 50μg/ml ZnO NPs. The CLSM images reveal the absorption and localization of ZnO NPs in cytoplasm and nuclei. The TOF-SIMS images demonstrate elevated levels of intracellular ZnO concentration and associated Zn concentration-dependent (40)Ca/(39)K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled (68)ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, (40)Ca/(39)K ratio, phosphocholine fragments, and glutathione fragments. The trend of change in TOF-SIMS spectra and images of ZnO NPs treated HaCaT cells demonstrate the possible mode of actions by ZnO NP involves cell membrane disruption, cytotoxic response, and ROS mediated apoptosis.
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Pei-Ling
authors.initials: PL
authors.lastname: Lee
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Bo-Chia
authors.initials: BC
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Gollavelli
authors.affiliation: Graduate Institute of Medical Science, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Sin-Yu
authors.initials: SY
authors.lastname: Shen
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Yu-Sheng
authors.initials: YS
authors.lastname: Yin
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Shiu-Ling
authors.initials: SL
authors.lastname: Lei
authors.affiliation: Department of Dermatology, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Cian-Ling
authors.initials: CL
authors.lastname: Jhang
authors.affiliation: Department of Dermatology, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Woan-Ruoh
authors.initials: WR
authors.lastname: Lee
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan; Graduate Institute of Medical Science, Taipei Medical University, Taipei 11031, Taiwan. Electronic address: ycling@mx.nthu.edu.tw.
authors.forename: Yong-Chien
authors.initials: YC
authors.lastname: Ling
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers / Q000737:chemistry*; D006801:Humans; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry*; D015203:Reproducibility of Results; D012867:Skin / Q000166:cytology / Q000187:drug effects*; D012995:Solubility; D018629:Spectrometry, Mass, Secondary Ion / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties; D018675:Toxicity Tests / Q000295:instrumentation / Q000379:methods; D015033:Zinc Isotopes; D015034:Zinc Oxide / Q000008:administration & dosage / Q000633:toxicity*
chemical_list: D004337:Drug Carriers; D015033:Zinc Isotopes; D015034:Zinc Oxide
keywords: HaCaT cells; Nanotoxicity; Stable isotope-labeled; TOF-SIMS; ZnO nanoparticles