Advances in the Mechanistic Understanding of Iron Oxide Nanoparticles' Radiosensitizing Properties

Abstract

Among the plethora of nanosystems used in the field of theranostics, iron oxide nanoparticles (IONPs) occupy a central place because of their biocompatibility and magnetic properties. In this study, we highlight the radiosensitizing effect of two IONPs formulations (namely 7 nm carboxylated IONPs and PEG₅₀₀₀-IONPs) on A549 lung carcinoma cells when exposed to 225 kV X-rays after 6 h, 24 h and 48 h incubation. The hypothesis that nanoparticles exhibit their radiosensitizing effect by weakening cells through the inhibition of detoxification enzymes was evidenced by thioredoxin reductase activity monitoring. In particular, a good correlation between the amplification effect at 2 Gy and the residual activity of thioredoxin reductase was observed, which is consistent with previous observations made for gold nanoparticles (NPs). This emphasizes that NP-induced radiosensitization does not result solely from physical phenomena but also results from biological events.

Keywords: X-ray irradiation; biological mechanism; cancer therapy; iron oxide nanoparticles; radiosensitization; thioredoxin reductase.

Figure 1

(A) TEM image of 7 nm carboxylated IONPs; (B) TEM image of 7 nm PEGylated IONPs, the scale bar corresponds to 100 nm. (C) Comparison of size distributions (histograms fitted with a log-normal function), obtained after statistical analysis of TEM images; (D) zeta potential measurements of each sample obtained by PCS; (E) size distributions of each sample obtained by PCS.

Figure 2

Iron concentrations in A549 cells assessed after 6 h, 24 h and 48 h of incubation with (A) 7 nm carboxylated IONPs and (B) 7 nm IONPs PEG₅₀₀₀ ([Fe] = 50 µg·mL⁻¹). Cellular iron content was quantified by the Perls’ Prussian blue stain method. Iron concentrations are expressed as mean values ± S.D for three independent experiments (Tukey test, ** p < 0.01, * p < 0.05, ns = not significant).

Figure 3

Confocal microscopy micrographs of iron oxide nanoparticle uptake in A549 cells and intracellular localization. Cells preincubated with 7 nm IONPs PEG₅₀₀₀ (left); 7 nm carboxylated IONPs (right) ([Fe] = 50 µg·mL⁻¹) for 48 h (scale bar: 20 μm). Red channel: Rhodamine-IONPs and Blue channel: LysoTracker Blue.

Figure 4

Survival fractions determined by standard clonogenic assay for A549 cells pre-incubated with and without IONPs ([Fe] = 50 μg·mL⁻¹) for (A) 6 h, (B) 24 h and (C) 48 h and irradiated by X-rays. Data are plotted as means ± SD from three independent experiments. One-way ANOVA analysis was performed for each result (Tukey test, ** p < 0.01, ns = not significant).

Figure 5

TrxR activity rates calculated from the slope of corresponding TrxR activity curves extracted from the measurement of absorption at 412 nm during 10 min for A549 cells treated with ([Fe] = 50 µg·mL⁻¹) and without 7 nm carboxylated IONPs and 7 nm IONPs PEG₅₀₀₀ for 6 h of incubation (A,D), 24 h of incubation (B,E) and 48 h of incubation (C,F). Data are plotted as means ± SD from three independent experiments. One-way ANOVA analysis was performed for each result (Tukey test, *** p < 0.001, ns = not significant).

Figure 6

The amplification factor at 2 Gy obtained in A549 cells as a function of residual TrxR activity. Gold data (blue) reported by Penninckx et al. [38] showed the influence of 10 nm amino-PEG GNPs on five different cell lines, while IONP data (red) showed the influence of two IONPs on A549 cells over time. Data are plotted as means ± SD from three independent experiments. Pearson’s correlation analysis was carried out for the scatterplot (r = −0.913).