Quantification of cell-associated Ag Nanoparticles and related ROS generation using Flow cytometry

Abstract

Silver nanoparticles (Ag NPs) are one of the most widely used among nanomaterials because of their antimicrobial activity. However, the use of Ag NPs is a subject still under debate, since Ag NPs are also known to cause cytotoxicity, which is related to intracellular reactive oxygen species (ROS) production. At certain high level concentration, ROS can cause serious cytotoxicity such as cell cycle damage, DNA damage and cell death.[1] When Ag NPs entered cells, Ag NPs stimulate organelles. In response to stimulation, ROS are produced actively. Therefore, to understand the ROS induction by cellular Ag NPs (c-Ag NPs), we need to know the amount of Ag NPs located within cells. In this study, we proposed an estimation method for the relationship between the c-Ag NPs and ROS production using flow cytometry (FCM).[2] Until now, to measure the uptake of Ag NPs in cells, various analytical methods have been used. Transmission electron microscope (TEM) were used to observe c-Ag NPs. Inductively coupled plasma mass spectrometry (ICP-MS) measurement was conducted to quantify c-Ag NPs. However, these approaches have some limitations such as applicability on various elements, complexity of pre-treatment. Therefore FCM was used as an alternative method to measure side scattering (SSC) intensity, which means cellular uptake, and fluorescent (FL) intensity, which is indicative of ROS production simultaneously. Experiments were performed on the lung cancer cell line, A549 exposed to Ag NPs with positive and negative charges. Both SSC and FL intensity increment was observed for the A549 cells. This simple quantitative estimation method can be helpful in predicting reaction between Ag NPs and biological fluids when nanomaterials are used inside human body for drug delivery or other medical purposes.