Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime...

Area of Science:

• Biomedical Science
• Nanotechnology
• Cellular Biology

Background:

• Nanoparticles offer therapeutic potential but can induce intracellular reactive oxygen species (ROS) overproduction, a key nanotoxicity mechanism.
• Existing methods for single-cell ROS detection lack sensitivity and high-throughput capabilities.

Purpose of the Study:

• To develop a sensitive and high-throughput single-cell method for detecting ROS.
• To investigate the nanotoxicity of gold nanoparticles by measuring low-level ROS generation.
• To correlate ROS levels with cellular damage and adverse effects.

Main Methods:

• Fluorescence Lifetime Imaging Microscopy for ROS detection (FLIM-ROX) was developed.
• FLIM-ROX utilizes a unique ROS reporter dye fluorescence lifetime to distinguish ROS signals from autofluorescence.
• Subcytotoxic amounts of cationic gold nanoparticles were applied to J774A.1 cells and primary human macrophages.

Main Results:

• FLIM-ROX detected low ROS levels induced by gold nanoparticles, which were undetectable by conventional methods.
• Gold nanoparticle exposure led to cellular morphology changes, elevated senescence, and DNA damage.
• Multiphoton FLIM-ROX allowed in vivo quantification of spatial ROS distribution in skin tissue.

Conclusions:

• FLIM-ROX is a sensitive and high-throughput method for detecting low-level ROS in vitro and in vivo.
• Low-level oxidative stress from nanoparticles is linked to cellular adverse effects and nanotoxicity.
• This method enhances the understanding of ROS-associated nanotoxicity.