Label-free cell based impedance measurements of ZnO nanoparticles—human lung cell interaction: a comparison with MTT, NR, Trypan blue and cloning efficiency assays

Abstract

Background There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. Results ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. Conclusions ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies. Graphic Abstract

Dually Modified Cellulose as a Non-Viral Vector for the Delivery and Uptake of HDAC3 siRNA

“…Within the first 24 h after stimulation, N/P ratio 20 induced an initial increase in resistance ( Figure 9 C) and a decrease in capacitance ( Figure S10C ). This effect of initial resistance increase has already been described for ECIS studies of bacterial infection and the cytotoxicity testing of nanoparticles [ 52 , 53 ]. However, the underlying mechanisms are not fully understood.…”

Binucleated cell formation and oncogene expression after particulate matter exposure is preceded by microtubule disruption, dysregulated cell cycle, prolonged mitosis, and septin binding

“…The effect of TiO2 NTs on microtubule organization suggests that the nanotubes might also affect other cellular processes where microtubules have a key role, such as cell signaling, proliferation, motility, and division, the phenomenon already observed after exposure to other metal oxide nanoparticles [41][42][43] . To quantify if the structural changes in microtubule organization are intrinsically connected to the potential changes in the cell division cycle, we conducted multiple time-lapse experiments on an expanded and stitched FoV on all studied nanomaterials and the control.…”

Nanostructured Medical Devices: Regulatory Perspective and Current Applications

“…Also, the pro-mitogenic activity of NPs should be considered, as shown in reference [18] for ZnO NPs at low concentrations. In particular, the presence of ZnO NPs was shown to stimulate pro-mitotic levels of cyclin B1.…”

“…According to Art. 2 of the MDR, the following definitions apply: "(18) 'nanomaterial' means a natural, incidental or manufactured material containing particles in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1-100 nm; Fullerenes, graphene flakes and single-wall carbon nanotubes with one or more external dimensions below 1 nm shall also be deemed to be nanomaterials; (19) 'particle', for the purposes of the definition of nanomaterial in point (18), means a minute piece of matter with defined physical boundaries;…”

“…(20) 'agglomerate', for the purposes of the definition of nanomaterial in point (18), means a collection of weakly bound particles or aggregates where the resulting external surface area is similar to the sum of the surface areas of the individual components;…”