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Dispersed single wall carbon nanotubes do not impact mitochondria structure or function, but technical issues during
Brian D Holt1, Vera Roginskaya, Bennett Van Houten
1Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213-3815, USA.
Journal of Materials Chemistry. B
|April 9, 2020
Summary
Single-wall carbon nanotubes (SWCNTs) interfere with standard assays measuring mitochondrial function due to surface activity and fluorescence quenching. Luciferase assays confirm SWCNTs do not impact ATP levels, highlighting their utility for nanomaterial studies.
Area of Science:
- Biotechnology
- Nanomaterial Science
- Cell Biology
Background:
- Mitochondria are vital organelles responsible for cellular energy production (ATP) and apoptosis.
- Understanding nanomaterial interactions with organelles like mitochondria is crucial for assessing cellular toxicity and function modulation.
- Single-wall carbon nanotubes (SWCNTs) are nanomaterials with potential applications requiring knowledge of their organelle interactions.
Purpose of the Study:
- To investigate the impact of SWCNTs, dispersed with Pluronic F127 and protein, on mitochondrial function.
- To evaluate the reliability of standard cellular assays when used with SWCNTs.
- To identify reliable methods for studying nanomaterial effects on mitochondria.
Main Methods:
- Seahorse XF24 analysis to assess mitochondrial respiration.
- Mitochondrial functional dye JC-1 imaging to evaluate membrane potential.
- Co-localization studies using microscopy.
- Light-emitting (luciferase) assays to measure ATP levels.
Main Results:
- Seahorse XF24 data indicated artifactual loss of mitochondrial function due to SWCNT probe adsorption.
- JC-1 imaging yielded inconclusive results due to SWCNT-induced fluorescence quenching.
- No significant co-localization or reorganization of mitochondria with SWCNTs was observed, despite potential misinterpretation without accounting for quenching.
- Luciferase assays demonstrated that SWCNTs do not alter cellular ATP levels.
Conclusions:
- The surface activity and fluorescence quenching properties of SWCNTs significantly interfere with traditional cellular assays.
- Standard assays like Seahorse and JC-1 imaging are unreliable for studying SWCNT effects on mitochondria.
- Light-emitting assays provide a quantitative and unambiguous method for assessing nanomaterial impact on cellular energy metabolism, confirming mitochondrial function is not affected by SWCNTs.
