Evaluation of Viability and Proliferation Profiles on Macrophages Treated with Silica Nanoparticles In Vitro via Plate-Based, Flow Cytometry, and Coulter Counter Assays
Nanoparticles (NPs) are known to interfere with many high-throughput cell viability and cell proliferation assays, which complicates the assessment of their potential toxic effects. The aim of this study was to compare viability and proliferation results for colloidal silica (SiO2 NP; 7？nm) in the RAW 264.7 mouse macrophage cell line using three different techniques: plate-based assays, flow cytometry analysis, and Coulter counter assays. Our data indicate that CellTiter-Blue, XTT, and CyQuant plate-based assays show increased values over control at low SiO2 NPs concentrations (0.001–0.01？g/L). SiO2 NPs show little-to-no interference with flow cytometry and Coulter counter assays, which not only were more reliable in determining cell viability and proliferation at low concentrations in vitro, but also identified changes in cell granularity and size that were not captured by the plate-based assays. At high SiO2 NP concentrations (1？g/L) all techniques indicated cytotoxicity. In conclusion, flow cytometry and Coulter counter identified new cellular features, and flow cytometry offered more flexibility in analyzing the viability and proliferation profile of SiO2 NP-treated RAW 264.7 cells. 1. Introduction Nanoparticles (NPs) have been proposed as promising tools for therapy, drug delivery, imaging, and active pharmaceutical ingredients [1–5]. An early and crucial stage in drug development is toxicity testing. High throughput in vitro toxicity assays that are widely employed for small molecule drug screening include assays for cell membrane integrity, oxidative stress, apoptosis, proliferation, or metabolic activity [6]. These assays have also been used for NPs toxicity evaluation [7–10]. Although plate-based assays offer several advantages (e.g., fast, easy, and reproducible), accumulating data show that NP interference with plate-based assay substrates can lead to erroneous data and provide little correlation with in vivo studies [11–14]. Some of NP properties that were reported to interfere with viability plate-based assays are surface charge, agglomeration/aggregation, hydrophobicity, and optical or magnetic properties [15–17]. For example, it was reported that titanium dioxide NPs (TiO2 NP; various size and concentrations) bind lactate dehydrogenase (LDH, indicator of cell viability) and alter assay readout [18]; copper NPs (Cu NP, 40？nm) and silver NPs (Ag NP, 35？nm) inactivate LDH [19]; gold NPs (Au NP; 10？nm) can absorb and traffic amine-containing dyes inside cells resulting in false positive results for membrane permeability assays [11];
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