Nanomaterial cytotoxicity is composition, size, and cell type dependent

Abstract

Background: Despite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs]) with differing size (MWCNTs of different diameters < 8 nm, 20-30 nm, > 50 nm; but same length 0.5-2 microm) to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT) bronchiolar epithelial cells.

Results: Following characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination.

Conclusions: Nanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the target cell type are critical determinants of intracellular responses, degree of cytotoxicity and potential mechanisms of toxicity.

Figure 1

Nanoparticle mediated protein depletion from cell culture media. Nanoparticles were incubated in serum containing culture media (10%) for periods of 30 minutes and 2 hours, with the early time point representing time for significant protein interaction and the later time point representing onset of particle uptake. Initially, all nanoparticles adsorb approximately 30% of the serum from the media, followed by a period of particle dependent reversible exchange of protein with culture media (a). Plotting of change in protein depletion from the media reveals significant differences in exchange based on nanoparticle properties (b). Vertical lines denote ±1 SD (n = 4 for all tested samples). (*) indicates significant t-test at (P < 0.05). Bracket with a (*) represents significant one-way ANOVA at (P < 0.05).

Figure 2

Analysis of nanomaterial uptake. Nanomaterial uptake by (a) 3T3, (b) hT, and (c) RAW cells. Unexposed cells were scanned to establish normal cell population. Cells exposed to nanoparticles for 3 hrs were detached from well plates and analyzed for forward scatter vs. side scatter to determine nanoparticle uptake. In the case of nanomaterial exposed cells, nanomaterial uptake/surface adsorption is reflected by increases in the side scatter of cell populations.

Figure 3

Nanomaterial composition, concentration, and size effects on 3T3, hT and RAW cells. The effect of nanomaterial concentration and composition on survival rates of (a) 3T3 fibroblasts, (b) hT, and (c) RAW cells at 24 hours. Cells were respectively treated with 10, 100, 1000 μg/ml of TiO2, SiO2, MWCNT <8 nm, MWCNT 20-30 nm, and MWCNT >50 nm for 24 hours. Viability was measured using the MTS assay and normalized to untreated cells. Vertical lines denote ±1 SD (n = 4 for all tested samples). Significant ANOVA test among different cell types is represented by bracket with a (*) over each data set. (*) above nanomaterial columns indicates statistically significant difference compared to untreated controls (P < 0.05).

Figure 4

Kinetics of cell dependent nanomaterial cytotoxicity. Time dependent toxicity profiles for each nanomaterial group. Nanomaterials cause time-dependent drops in cell viability. Nanomaterials at 100 μg/ml were exposed to (a) 3T3, (b) hT, and (c) RAW cells for 6, 12, 18, and 24 hours. Viability was measured using MTS assay and normalized to untreated cells. Bracket with a (*) represents significant one-way ANOVA at (P < 0.05).

Figure 5

Nanomaterial dependent ROS generation and lysosomal membrane destabilization (LMD) in 3T3, hT, and RAW cells. (a) ROS generation was quantified using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) for untreated cells and compared to cells exposed with each test nanomaterial for 2 hours at 100 μg/ml. LMD was visualized via acridine orange staining in (b) 3T3, (c) hT, and (d) RAW cells exposed to nanomaterials at 100 μg/ml for 4 hrs. In control (healthy) cells, lysosomes can be seen as red-orange granules and cytoplasm has a diffuse green fluorescence. In cells with lysosomal membrane damage, lysosomes exhibit a shift from red-orange to a green color and overall intensity of green fluorescence is increased in these cells. (e) Quantification of LMD after 4 hours of exposure to nanomaterials at 100 μg/ml was done via acridine orange relocation technique. For each nanomaterial, bracket with a (*) indicates significant difference between different cell types by ANOVA. (*) above nanomaterial columns indicates significant difference from untreated cells by Bonferroni test.

Figure 6

Nanomaterial dependent Mitochondrial membrane potential (MMP) and Caspase-3/7 activation in 3T3, hT, and RAW cells. (a) MMP was monitored following 6 hrs of nanomaterial exposure at 100 μg/ml using MitoProbe DilC₁(5) Assay Kit. (b) Caspase activity in all three cell types was assessed using Sensolyte Homogeneous AMC Caspase-3/7 Assay Kit following 10 hours of exposure to all materials at 100 μg/ml. For each nanomaterial, bracket with a (*) indicates significant difference between different cell types by ANOVA. (*) above nanomaterial columns indicates significant difference from untreated/control cells by Bonferroni test.

Figure 7

Nanomaterial induced apoptosis and necrosis in 3T3, hT and RAW cells. Annexin V-FITC/PI double staining was performed to distinguish 3T3, hT and RAW cells undergoing (a) Apoptosis and (b) necrosis following 20 hours of exposure to all nanomaterials at 100 μg/ml. For each nanomaterial, bracket with a (*) indicates significant difference between different cell types by ANOVA. (*) above nanomaterial columns indicates significant difference from untreated/control cells by Bonferroni test.