Nanomaterials are organic or inorganic particles whose size is less than 100 nanometers in at least one dimension. Though nanomaterials hold tremendous promise for myriad applications, their impact on living cells in the short and long term are incompletely understood. Numerous studies have suggested that nanomaterials, even when made of inert materials, can still display toxicity because of their physical properties (size, shape). Moreover, nanomaterials have been shown to cause oxidative stress, cytokine production, and cell death. Understanding the multifaceted cytotoxicity of nanoparticles is thus important to public health. A challenge in characterizing the effect of nanoparticles on cells is that – because of their high adsorption capacity, high optical activity, redox potential, and chemical reactivity – some particle types interfere with conventional techniques for assessing cell viability/health (Figure 1).
Figure 1. Nanoparticles can interfere with traditional toxicity/viability assays. The high adsorption capacity, high optical activity, redox potential, and/or chemical reactivity of certain types of nanoparticles make them incompatible with traditional cell viability assays.
As an alternative, label-free means of evaluating nanoparticle induced cytotoxicity, impedance-based xCELLigence real-time cell analysis (RTCA) has proven to be extremely effective. In a study by Scott Boitano and colleagues at the University of Arizona the potential cytotoxicity of 11 different inorganic nanomaterials (Ag0, Al2O3, CeO2, Fe0, Fe2O3, HfO2, Mn2O3, SiO2, TiO2, ZnO, and ZrO2) was evaluated using the 16HBE14o- human bronchial epithelial cell line. The effect of these different materials varied greatly (only four are shown here). Whereas Mn2O3 was strongly cytotoxic, SiO2 and Al2O3 were moderately toxic, and CeO2 had no effect at the concentrations tested (Figure 2).
An important question is whether the results obtained by RTCA correlate with results obtained using orthogonal techniques. As seen in Figure 3, data from a side-by-side MTT end point assay correlates extremely well with the RTCA assay. This publication and others like it have firmly established that the accuracy and reproducibility of RTCA assays, coupled with the reduced work load and continuous data acquisition (no data points are “missed”) make the xCELLigence system an excellent means of evaluating nanotoxicity.
Figure 2. Real-time impedance monitoring of nanoparticle-induced cytotoxicity. At the ~17 hour time point (denoted by the dashed vertical line) 16HBE14o- human bronchial epithelial cells were exposed to different concentrations of the indicated nanoparticles, and impedance was monitored continuously for the next ~60 hours. (A) Nano-CeO2 concentrations (mg/L): 0 (—), 250 (— • —), 500 (- – -), and 1000 (•••). (B) Nano-Al2O3 concentrations (mg/L): 0 (—), 250 (— • —), 500 (- – -), and 1000 (•••). (C) Nano-SiO2 concentrations (mg/L): 0 (—), 100 (— • —), 200 (- – -), 300 (•••), and 600 (— —). (D) Nano-Mn2O3 concentrations (mg/L): 0 (—), 10 (— • —), 20 (- – -), 50 (•••), and 100 (— —).
Figure 3. Comparison of cytotoxicity measurements made using xCELLigence (•) vs. MTT (○) assay. Percent of response (relative to untreated control) is plotted as a function of nanoparticle concentration.
Key Benefits of Using xCELLigence for Studying Nanotoxicity:
- Impedance detection method is not interfered with by nanoparticles.
- Continuous monitoring ensures no meaningful data points are missed.
- Real-time data allows identification of the optimal times for treatment and data collection.
- Non-invasive assay is performed in tissue culture incubator, allowing for analysis by orthogonal standard viability assays at any point during the experiment.
- Easy quantification of the onset and kinetics of the cytotoxic response.