Cytotoxicity of cadmium-containing silica nanoparticles Cd-SiO2NPs (0.05–100？μg/mL) versus SiO2NPs and CdCl2 was evaluated by an in vitro test battery in A549 by assessing (i) mitochondrial function, (ii) membrane integrity/cell morphology, (iii) cell growth/proliferation, (iv) apoptotic pathway, (v) oxidative stress, after short- (24–48？h) and long-term (10 days) exposure. Both Cd-SiO2NPs and CdCl2 produced dose-dependent cytotoxic effects: (i) MTT-assay: similar cytotoxicity pattern was observed at both 24 and 48？h, with a more Cd-SiO2NPs pronounced effect than CdCl2. Cd-SiO2NPs induced mortality (about 50%) at 1？μg/mL, CdCl2 at 25？μg/mL; (ii) calcein-AM/PI staining: decrease in cell viability, noticeable at 25？μg/mL, enhanced markedly at 50 and 100？μg/mL, after 24？h. Cd-SiO2NPs induced higher mortality than CdCl2 (25% versus 4%, resp., at 25？μg/mL) with further exacerbation after 48h; (iii) clonogenic assay: exposure for longer period (10 days) compromised the A549 proliferative capacity at very low dose (0.05？μg/mL); (iv) a progressive activation of caspase-3 immunolabelling was detected already at 1？μg/mL; (v) GSH intracellular level was modified by all compounds. In summary, in vitro data demonstrated that both Cd-SiO2NPs and CdCl2 affected all investigated endpoints, more markedly after Cd-SiO2NPs, while SiO2NPs influenced GSH only. 1. Introduction The rapid development of nanotechnology worldwide is accompanied by massive generation and usage of engineered nanoparticles (ENPs), even though essentially most of these NPs have not been sufficiently examined for potential toxicity at this time [1, 2]. Thus, with the exponential growing production of ENPs, the potential for the respiratory system to be exposed to a seemingly countless number of unique NPs is expected to increase, and many aspects related to the size of these nanomaterials, smaller than cells and cellular organelles, have raised concerns about safety [2–4]. Among ENPs, silica/cadmium containing nanomaterials have attracted much attention in the latest years for their applications in medicine and industrial manufacturing, synthesis, and engineering [5–10]. Though silica nanoparticles (SiO2NPs) are generally considered to be nontoxic, experiments using cell cultures or animal models have indicated dose-dependent cytotoxicity, increased reactive oxygen species, and reversible lung inflammation [11–19]. On the other hand, a large body of evidence supports lung toxicity effects after cadmium exposure when inhaled [20, 21], and although its toxicity mechanisms are not yet fully understood,
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