Silica-Coated SPIONs Induce Ferroptosis in Endothelial Cells While Oleic Acid Mitigates Their Cytotoxic Effects

Abstract

Purpose: Induction of ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, holds promise as a novel cancer therapy. Superparamagnetic iron oxide nanoparticles (SPIONs) have been proven able to induce ferroptosis in tumour cells, while their effects on non-cancerous cells remain unclear. In this study, we investigated the ability of silica-coated SPIONs to induce ferroptosis in human umbilical vein endothelial cells (HUVEC) and explored the potential protective effects of oleic acid (OA). Additionally, we evaluated the applicability of scanning electron microscopy (SEM) in distinguishing between ferroptotic and apoptotic cell death.

Results: We confirmed that silica-coated SPIONs, (used at concentrations of 25 and 50 µg/mL) increased lipid peroxidation and ROS formation in a dose-dependent manner up to 4.9- and 4-fold compared to controls, ultimately promoting ferroptosis without evidence of apoptosis, as indicated by the absence of phosphatidylserine-positive, propidium iodide-negative cells in flow cytometry experiments. Consistent with these results, the ferroptosis inhibitors α-tocopherol and ferrostatin-1 attenuated SPION-induced cytotoxicity, supporting ferroptosis as the primary mechanism of cell death. OA also protected cells from SPION-induced cytotoxicity by reducing lipid peroxidation, ROS formation, and cell death (from 58% to 26%), while increasing glutathione peroxidase expression. Unfortunately, due to the similar surface morphology of ferroptotic and apoptotic cells, SEM is not a reliable method for distinguishing between these two forms of cell death.

Conclusion: This study provides important insights into the mechanisms of toxicity of silica-coated SPIONs in endothelial cells and highlights the potential role of OA as a modulator of SPION-induced side effects.

Keywords: GPX4; ROS; SPION; ferroptosis; lipid peroxidation; oleic acid.

Figure 1

Nanoparticle characterization. Zeta potential distribution of silica-coated superparamagnetic iron oxide nanoparticles (SPIONs) in serum-free medium (A), hydrodynamic size distribution and polydispersity index of silica-coated SPIONs in serum-free medium (B), and representative transmission electron microscopy (TEM) images of as-synthesized SPIONs (C), and silica-coated SPIONs (D).

Figure 2

Silica-coated SPIONs increased lipid peroxidation in HUVEC cells in a concentration-dependent manner. Human umbilical vein endothelial cells (HUVEC) were stained with BODIPY 581/591 C11 dye (BODIPY), a lipid peroxidation indicator, and exposed to various concentrations of silica-coated superparamagnetic iron oxide nanoparticles (SPIONs) for 24 hours and then analysed by flow cytometry. The data are presented as follows: (A) Representative flow cytometry histograms showing the fluorescence emission of oxidized (OX) and reduced (R) BODIPY in untreated control cells (light blue) and in cells treated with 25 µg/mL (orange) or 50 µg/mL (red) SPIONs. (B) Quantification of lipid peroxidation, expressed as fold change of the OX/R BODIPY geometric mean fluorescence intensity (MFI) ratio in SPION-treated cells versus the same ratio calculated in untreated control cells (± SEM). Data represent the mean of at least three independent experiments. Statistical significance is indicated as: ***P < 0.001; ****P < 0.0001.

Figure 3

Ferroptosis inhibitors decrease lipid peroxidation, oxidative stress, and cell death caused by silica-coated SPIONs. To quantify ROS production and cell death (A and B), HUVEC were exposed to SPIONs and/or selected ferroptosis inhibitors or oleic acid for 24 hours and then stained with CM-H₂DCFDA and 7-AAD dyes and analysed via flow cytometry. Data are presented either as % of 7-AAD positive cells or as fold change of the CM-H₂DCFDA MFI signal calculated versus the MFI of control cells. To measure lipid peroxidation instead (C), cells were first stained with the lipid peroxidation sensor, BODIPY, and then exposed to silica-coated SPIONs and/or selected ferroptosis inhibitors or oleic acid for 24 hours and subsequently analysed via flow cytometry. The data in the graphs show the ratio of the MFI of the oxidized (OX) and reduced (R) BODIPY dye of a specific sample normalized by the same ratio calculated in untreated control cells. Bars show mean values (± SEM) calculated from at least three independent experiments. Only statistical significance within the same sample ± ferroptosis inhibitors or oleic acid is shown (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 4

Silica-coated SPIONs induce ferroptosis via decreasing GPX4 expression. HUVEC were seeded in 6-well plates and exposed to oleic acid, selected ferroptosis inductors or inhibitors and/or silica-coated SPIONs (50 µg/mL) for 24 hours. After 24 hours, cells were lysed, proteins in the cell lysates were separated by polyacrylamide gel electrophoresis in the presence of SDS, and Western blotting was performed, followed by immunodetection of GPX4 and β-actin proteins on a nitrocellulose membrane (A). The spots on the membrane were analysed densitometrically using ImageJ. Data in the graph shows the ratio of GPX4 to β-actin protein band intensities normalized to their corresponding values in unexposed cells. In (B) are shown the mean values of four biological replicates of the experiment (± SEM). Values that are statistically significantly different from controls or a specified sample are indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 5

Silica-coated SPIONs do not induce PS exposure. Cells were exposed to ferroptosis inducer RSL3, apoptosis inducer staurosporine and silica-coated SPIONs for 24 hours and then double stained with Annexin V-Pacific blue and PI. The cells were then analysed using a flow cytometer. The dot plots (left) show the cell population identified by FCS and SSC selected for further analysis of PS exposure and cell viability, which is shown in the contour plots (right). The lower left quadrant represents double-negative cells that are viable and do not have exposed PS, the lower right quadrant represents Annexin V-positive and PI-negative cells with intact cell membrane and exposed PS, and the upper right quadrant represents double-positive cells that have lost cell membrane integrity.

Figure 6

SEM micrographs of cells exposed to staurosporine, RSL3, and SPIONs.

Figure 7

TEM micrographs of untreated cells and cells exposed to SPIONs. (A–C) images of untreated cells. (D–H) images at different magnifications of cells undergoing ferroptosis in response to the treatment with 50 µg/mL SPIONs.