Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering

Abstract

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe3O4/SiO2 NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe3O4/SiO2 NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe3O4/SiO2 NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.

Figure 1. Characterization of bare Fe₃O₄/SiO₂ NPs in water.

(A) TEM image, (B) Dynamic light scattering, (C) ζ-Potential measurements.

Figure 2. Characterization of bare Fe₃O₄/SiO₂ NPs in cell culture medium.

(A) ζ-potential and (B) Dynamic light scattering after 96 h incubation in DMEM culture medium.

Figure 3. Effect of bare and passivated Fe₃O₄/SiO₂ NPs on the viability and membrane damage in two cell lines (A549 and HeLa).

(A, B) WST-8 proliferation assay and (C, D) LDH assay on A549 and HeLa cells incubated with increasing concentrations (0.5, 1, 2.5, 5 nM) of bare and passivated Fe₃O₄/SiO₂ NPs at different times (48 and 96 h). C identifies the negative control in the absence of NPs. Viability of NPs-treated cells is expressed relative to non-treated control cells. As positive control (P), cells were incubated with 5% DMSO in WST-8 assay and 0.9% Triton X-100 in LDH assay (not shown). Data are reported as mean ±SD from three independent experiments; *P < 0.05 compared with control (n =  8).

Figure 4. Effect of bare and passivated Fe₃O₄/SiO₂ NPs on the ROS level in two cell lines (A549 and HeLa).

Cells were treated with different concentration (0.1, 0.5, 1, 2.5 nM) of NPs for 48 and 96 h and ROS levels were evaluated by DCFH-DA assay. Percent ROS generation of nanoparticle-treated cells is expressed relative to non-treated control cells. As a positive control (P), cells were incubated with 500 µM H₂O₂ (not shown). Data are reported as mean ± SD from three independent experiments; *P < 0.05 compared with control (n =  8).

Figure 5. Effect of bare and passivated Fe₃O₄/SiO₂ NPs on mitochondrial membrane potential (MMP) in two cell lines.

Cells were treated with 2.5 nM of NPs for 48 h and percent of mitochondrial membrane potential of nanoparticle-treated cells was evaluated by JC-1 assay and was expressed relative to non-treated control cells. As a positive control (P) cells were incubated with 100 µM valinomycin. Data are reported as mean ± SD from three independent experiments; *P < 0.05 compared with control (n =  8).

Figure 6. Effect of bare and passivated Fe₃O₄/SiO₂ NPs on DNA damage.

A549 and HeLa cells were treated with 2.5 nM of NPs for 48 h. DNA damage was evaluated through the comet assay by A) tail length and B) tail DNA intensity. Values shown are mean from 100 randomly selected comet images of each sample. As a positive control (P) cells were incubated with 500 µM H₂O₂. Data are reported as mean ± SD from three independent experiments; *P < 0.05 compared with control (n =  3).

Figure 7. Cell uptake of bare and passivated Fe₃O₄/SiO₂ NPs.

Normalized internalization data for A549 and HeLa cells expressed as the number of nanoparticles internalized (determined by ICP-AES) per cell after 48 and 96 h of NPs incubation. Data are reported as mean ± SD from three independent experiments.

Figure 8. Effects of pH and surface functionalization on iron ions release from bare and passivated Fe₃O₄/SiO₂ NPs.

NPs degradation was evaluated both at pH 4.5 and pH 7 from 3 to 96 h. Neutral conditions were also probed in cell culture medium (DMEM, 10% FBS, pH 7.4), obtaining the same results (i.e., no detectable ion release).

Figure 9. Effect of the iron chelator desferioxamine (DFX) on A549 and HeLa cells treated with NPs.

Cells were treated with 100 µM of DFX and 5 nM of passivated NPs (positive and negative) or 2.5 nM of bare NPs for 96 h. Data are reported as mean ± SD from three independent experiments; *P < 0.05 compared with control (n =  8).

Effect of Fe₃O₄/SiO₂ NPs surface passivation on the viability in two cell lines (A549 and HeLa).

A) Viability of A549 and HeLa cells after 96h exposure to 2.5 nM of Fe₃O₄/SiO₂ NPs with different amount of amine groups with WST-8 assay. Percent viability of nanoparticle-treated cells is expressed relative to non-treated control cells. As positive control (P), cells were incubated with 5% DMSO. Data are reported as mean ± SD from three independent experiments; *P < 0.05 compared with control (n =  8). B) Effect of NPs surface passivation on iron ions release from Fe₃O₄/SiO₂ NPs. NPs degradation was evaluated both at pH 4.5 and 7 from 3 to 96 h.