Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells

Pei Ling Lee, Bo Chia Chen, Ganesh Gollavelli, Sin Yu Shen, Yu Sheng Yin, Shiu Ling Lei, Cian Ling Jhang, Woan Ruoh Lee, Yong Chien Ling

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 40Ca/39K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled 68ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, 40Ca/39K 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.

Original languageEnglish
Pages (from-to)3-12
Number of pages10
JournalJournal of Hazardous Materials
Volume277
DOIs
Publication statusPublished - Jul 30 2014

Fingerprint

Secondary Ion Mass Spectrometry
Zinc Oxide
imaging method
Cytotoxicity
Secondary ion mass spectrometry
Zinc oxide
Confocal Microscopy
Nanoparticles
microscopy
Microscopic examination
mass spectrometry
laser
zinc
oxide
Scanning
Imaging techniques
ion
Lasers
skin
Skin

Keywords

  • HaCaT cells
  • Nanotoxicity
  • Stable isotope-labeled
  • TOF-SIMS
  • ZnO nanoparticles

ASJC Scopus subject areas

  • Waste Management and Disposal
  • Environmental Chemistry
  • Environmental Engineering
  • Health, Toxicology and Mutagenesis
  • Pollution
  • Medicine(all)

Cite this

Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells. / Lee, Pei Ling; Chen, Bo Chia; Gollavelli, Ganesh; Shen, Sin Yu; Yin, Yu Sheng; Lei, Shiu Ling; Jhang, Cian Ling; Lee, Woan Ruoh; Ling, Yong Chien.

In: Journal of Hazardous Materials, Vol. 277, 30.07.2014, p. 3-12.

Research output: Contribution to journalArticle

Lee, Pei Ling ; Chen, Bo Chia ; Gollavelli, Ganesh ; Shen, Sin Yu ; Yin, Yu Sheng ; Lei, Shiu Ling ; Jhang, Cian Ling ; Lee, Woan Ruoh ; Ling, Yong Chien. / Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells. In: Journal of Hazardous Materials. 2014 ; Vol. 277. pp. 3-12.
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AU - Chen, Bo Chia

AU - Gollavelli, Ganesh

AU - Shen, Sin Yu

AU - Yin, Yu Sheng

AU - Lei, Shiu Ling

AU - Jhang, Cian Ling

AU - Lee, Woan Ruoh

AU - Ling, Yong Chien

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AB - 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 40Ca/39K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled 68ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, 40Ca/39K 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.

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