Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture

Abstract

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

Keywords: 3D cell culture; PEG; cytotoxicity; dextran; endothelial cells; reactive oxygen species; superparamagnetic iron oxide nanoparticles.

Figure 1

5 and 30 nm nanoparticles were coated with dextran and polymer polyethylene glycol (PEG). Nanoparticle samples were sonicated for 1 h with either dextran or m-PEG-silane and imaged by TEM.

Figure 2

Iron oxide nanoparticles were taken into cells after 3 h incubation. (A) Porcine aortic endothelial cells (PAEC) were incubated with 0.1 mg/mL bare, dextran or PEG coated iron oxide nanoparticles. Large nanoparticle aggregation was observed in vesicles in the cytoplasm; (B) Intracellular iron increased with nanoparticle concentration, as measured by iron absorbance. ^* p < 0.01 compared to 0.1 mg/mL for same coating condition, # p < 0.05 compared to 0.1 mg/mL for same coating condition.

Figure 3

Dextran and PEG coating reduced iron oxide nanoparticle cytotoxicity, as measured by a Live/Dead assay. PAEC were incubated with 0, 0.1, 0.25 and 0.5 mg/mL of 5 and 30 nm bare and coated nanoparticles for 24 h. (A) Selected fluorescent images in which green = live cells and red = dead cells; and (B) Quantification of dead cell number by Image J. ^* p < 0.01, # p < 0.05.

Figure 4

Bare 30 nm nanoparticles induced the highest level of intracellular ROS formation. (A) Selected confocal microscopy ROS images. PAEC were incubated with different concentrations of bare and coated nanoparticles for 3 h. Cells were then labeled with 10 μM carboxy-H₂DCFDA (green), a general ROS indicator. Scale bar = 100 μm; (B) Quantification of ROS formation by Image J. # p < 0.05.

Figure 5

Dextran and PEG coated nanoparticles reduced cell elongation and stress fiber formation. (A) Selected confocal images of cells labeled for actin. PAEC were incubated with 0.5 mg/mL bare and coated 5 and 30 nm nanoparticles for 24 h. Samples were then fixed with paraformaldehyde, permeabilized with Triton X-100, and labeled with rhodamine phalloidin (actin, red) and Hoechst (nuclei, blue). Scale bar = 20 μm; (B) Cell length was quantified by Image J. # p < 0.05.

Figure 6

Dextran and PEG nanoparticle coating improved cell viability in 3D culture. (A) Selected confocal images of PAEC viability in 3D alginate constructs with nanoparticles in the alginate, as measured by the Live/Dead assay. Alginate was mixed with 0.1 mg/mL bare and coated nanoparticles and cells. Alginate-nanoparticle-cell constructs were labeled using a Live/Dead assay. Cell viability in 3D constructs with (B) nanoparticles in the alginate and (C) nanoparticles inside cells was measured using an Alamar blue assay.