Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals

Abstract

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3-24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.

Keywords: MRI-optical dual imaging; SPIONs; cellular uptake; iron oxide; magnetic nanoparticles; multifunctional cancer diagnostics; optical biopsy of tissues cells.

Figure 1

Topographic atomic force microscopy (AFM) image of Fe₃O₄ magnetic nanoparticles dispersed on mica surface (B), particle height histogram (A), hydrodynamic size distribution (C) and schematic picture (D) of Fe₃O₄ magnetic nanoparticles.

Figure 2

Fixed NIH3T3 cells after 0.5–72 h of incubation with 65 ng/mL of Fe₃O₄ (stained with Prussian Blue) (A–F). The accumulation was observed using bright-field microscope equipped with digital color camera.

Figure 3

(A) viability of mouse embryonic fibroblasts NIH3T3, incubated with SPIONs for 3, 24 and 48 h. Toxicity of nanoparticles was investigated using XTT cell viability assay; (B) XTT plate images of NIH3T3 cells incubated with 32.5 ng/mL of SPION nanoparticles for 0, 24 and 48 h.

Figure 4

(A) T2W MR coronal slice images in vitro of different concentrations of Fe₃O₄ dissolved in water (from c1 (1300 mg/L) to c15 (13 mg/L)); (B) T2W MR signal intensity plot of aqueous suspensions of Fe₃O₄ versus the concentration in the solution. Dotted line marks water MR signal.

Figure 5

T2W MR coronal slice images of injection site at different time moments after intramuscular injection at dose of (A) 520 µg Fe₃O₄/kg (in the upper figure) and (B) 20.8 µg Fe₃O₄/kg (in the lower figure) in the rat left hind paw (red circles). The arrows marks injection site of physiological saline and Fe₃O₄ nanoparticles solution.

Figure 6

Relative MR signal intensity at the injection site versus time at the different doses of Fe₃O₄ (red curve—520 µg Fe₃O₄/kg, black—20.8 µg Fe₃O₄/kg).