Figure S2. (IONs) are frequently used in numerous biomedical applications, in particular as magnetic resonance imaging contrast agents in liver imaging. Indeed, quantity of IONs have been withdrawn because of the poor clinical overall performance. Yet comprehensive understanding of their relationships with hepatocytes remains relatively limited. Here we investigated how iron oxide nanocubes (IO-cubes) and clusters of nanocubes (IO-clusters) impact distinct human being hepatic cell lines. The viability of HepG2, Huh7 and Alexander cells was concentration-dependently decreased after exposure to either IO-cubes or IO-clusters. We found related cytotoxicity levels in three cell lines induced by both nanoparticle formulations. Our data show that different manifestation levels of Bcl-2 predispose cell death signaling mediated by nanoparticles. Both nanoparticles induced rather apoptosis than autophagy in HepG2. Contrary, IO-cubes and IO-clusters result in unique cell death signaling events in Alexander and Huh7 cells. Our data clarifies the mechanism by which cubic nanoparticles induce autophagic flux and the mechanism of subsequent toxicity. These findings imply that the cytotoxicity of ION-based contrast agents should be cautiously considered, particularly in individuals with liver diseases. Probe Assay was used as an internal control. Data were analyzed using Excel and MaxStat Pro 3.6 programs. Manifestation of target gene was normalized to manifestation by using the 2?CT method while described in [61]. Statistical analysis Quantitative results are present as mean??SEM. The statistical significance of differences between the groups was identified using ANOVA Newman-Keuls test. All statistical analyses were performed using MaxStat Pro 3.6. Statistical significance was recognized if the tested value was smaller than 0.05 (*), 0.01 (**) or 0.001 (***). When multiple pairwise comparisons were performed, the Bonferroni correction was used to adjust the significance level. For quantitative fluorescence microscopy analysis (analysis of lysosomal size, integrity, caspase-3 activity) rigorously defined guidelines for accuracy and precision quantification were used [62]. The sample size dedication was based on a statistical method explained in [63], which decides sample size for 95% confidence level and 0.8 statistical power equal to 15. Consequently, n?=?15 cells were used in quantification. Results Characterization of the nanoparticles IO-cubes and Vilanterol trifenatate IO-clusters were synthetized and functionalized as previously explained [64, 65]. The physicochemical properties of the IO-cubes and IO-clusters investigated with this study are summarized in Figs.?1 and ?and2.2. Transmission electron microscopy of both preparations exposed the same mean size of an iron oxide core of about 36 and Vilanterol trifenatate 38?nm Vilanterol trifenatate for IO-cubes and IO-clusters respectively (Fig.?1a and Additional file 1: Number S1). Accordingly, dynamic laser light scattering analysis in aqueous environment showed mean hydrodynamic diameters of about 140?nm for both IO-cubes and IO-clusters (Figs.?1b, ?b,2b).2b). The magnetization curves were related for IO-cubes and IO-clusters (Fig.?2a). Finally, XRD was used to confirm IO-cubes and IO-clusters structure and phase composition (Fig.?2c). Open in a separate windows Fig.?1 Physicochemical characterization of the iron oxide nanocubes (IO-cubes) and nanoclusters (IO-clusters). a Transmission electron micrographs of the iron core of the nanoparticles. b Physicochemical properties of IO-cubes and IO-clusters (hydrodynamic diameter, Rabbit Polyclonal to TCF2 saturation magnetization) Open in a separate windows Fig.?2 Physicochemical characterization of the iron oxide nanocubes (IO-cubes) and nanoclusters (IO-clusters). a Room heat magnetization curves of IO-cubes and IO-clusters using magnetic field range from ??30 to 30 kOe. b Hydrodynamic diameters of IO-cubes and IO-clusters in PBS after surface changes with PEG as measured by dynamic light scattering utilizing Zetasiser Nano ZS. c XRD patterns of IO-cubes and IO-clusters with the indexation of the Bragg peaks to an inverse spinel structure It is well worth noting here, that one of the widest areas of biomedical software of iron oxide nanoparticles is an MRI contrasting [28, 66]. Indeed, it was found that the size of nanoparticles between 50 and 200?nm seems to be the most effective for cellular uptake and MRI imaging applications [10, 28, 66, 67]. Particles having size similar with the diameter Vilanterol trifenatate of Vilanterol trifenatate liver sinusoidal fenestrations (up to 150C200?nm) have been shown to extravasate into the space.