Dynamic Monitoring of Metal Oxide Nanoparticle Toxicity by Label Free Impedance Sensing

Abstract

The increased use of nanoparticles in industrial and medical products is driving the need for accurate, high throughput in vitro testing procedures to screen new particles for potential toxicity. While approaches using standard viability assays have been widely used, there have been increased reports of the interactions of nanoparticles with their soluble labels or optical readouts which raise concerns over the potential generation of false positive results. Here, we describe the use of an impedance spectroscopy approach to provide real-time reagent free detection of toxicity for a panel of metal oxide nanoparticles (ZnO, CuO, and TiO(2)). Using this approach, we show how impedance measurements can be used to track nanoparticle toxicity over time with comparable IC(50) values to those of standard assays (ZnO-55 μg/mL, CuO-28 μg/mL) as well as being used to identify a critical 6 h period following exposure during which the nanoparticles trigger rapid cellular responses. Through targeted analysis during this response period and the use of a novel image analysis approach, we show how the ZnO and CuO nanoparticles trigger the active export of intracellular glutathione via an increase in the activity of the ATP dependent MRP/1 efflux pumps. The loss of glutathione leads to increased production of reactive oxygen species which after 2.5 h triggers the cells to enter apoptosis resulting in a dose dependent cytotoxic response. This targeted testing strategy provides comprehensive information beyond that achieved with standard toxicity assays and indicates the potential for cell-nanoparticle interactions that could occur following in vivo exposure.

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity, apoptosis, oxidative stress, and inflammation

“…Furthermore, in vitro glutathione levels have been reported to be very low to begin with, making GSH difficult to detect. Additionally, nanoparticles have been observed to trigger the active export of intracellular glutathione by increasing the activity of the ATP-dependent MRP/1 efflux pumps, which in return leads to increased ROS production and cellular apoptosis (Seiffert et al, 2012;Stone et al, 2009). Increased GSH levels induced by a number of NMs in the present study might be an indication of late oxidative stress reactions that occur as a detoxifying defense mechanism after the initial GSH decrease (Lin and Yang, 2007).…”

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines

“…Cell-based label-free technologies such as the xCELLigence RTCA system have been implemented as part of the preclinical drug development process [27-29] and also for the evaluation of the cytotoxicity of a variety of dissolved chemicals [30-33]. This technology was also used to assess the cytotoxicity of a variety of nanoparticles including quartz [34], cobalt ferrite [35], gold nanoparticles [36], quantum dots and gold nanorods [37], ZnO, CuO, and TiO 2 [38] and other inorganic nanoparticles [39]. …”

Zinc oxide nanoparticle and bovine serum albumin interaction and nanoparticles influence on cytotoxicity in vitro

“…More are known about the mechanisms of plain ZnO NPs actions: ZnO induce decrease in cell viability by inducing apoptosis, polymerisation of F-actin, ROS generation, reduced glutathione (GSH) depletions, and morphological changes in nucleus [36]. It was shown that Zn + ions at the maximum dissolution concentrations do not cause cytotoxicity, ZnO NPs do not disturb membrane integrity but activates multi-drug resistance pump MRP-1/PgP which export glutathione from the cell and can be a reason of apoptosis [37]. …”

“…It has been shown that oxidative stress plays a key role in the development of toxicity in response to nanoparticle challenge [36][37][38][39] and, in most cases, protein corona decreases ROS generation, characteristic to plain NPs [40]. Levels of ROS, generated by ZnO NPs in CHO cells are shown in Fig.…”