Induction of ferroptosis using functionalized iron-based nanoparticles for anti-cancer therapy

Abstract

Ferroptosis, a cell death pathway that is induced in response to iron, has recently attracted remarkable attention given its emerging therapeutic potential in cancer cells. The need for a promising modality to improve chemotherapy's efficacy through this pathway has been urgent in recent years, and this non-apoptotic cell death pathway accumulates reactive oxygen species (ROS) and is subsequently involved in lipid peroxidation. Here, we report cancer-targeting nanoparticles that possess highly efficient cancer-targeting ability and minimal systemic toxicity, thereby leading to ferroptosis. To overcome the limit of actual clinical application, which is the ultimate goal due to safety issues, we designed safe nanoparticles that can be applied clinically. Nanoparticles containing ferroptosis-dependent iron and FDA-approved hyaluronic acid (FHA NPs) are fabricated by controlling physicochemical properties, and the FHA NPs specifically induce ROS production and lipid peroxidation in cancer cells without affecting normal cells. The excellent in vivo anti-tumor therapeutic effect of FHA NPs was confirmed in the A549 tumor-bearing mice model, indicating that the induction of FHA NP-mediated cell death via the ferroptosis pathway could serve as a powerful platform in anticancer therapy. We believe that this newly proposed FHA NP-induced ferroptosis strategy is a promising system that offers the potential for efficient cancer treatment and provides insight into the safe design of nanomedicines for clinical applications.

Keywords: Biomaterials; Cancer nanomedicine; Fenton reaction; Ferroptosis; Iron-based nanoparticles; Reactive oxygen species.

Graphical abstract

Fig. 1

Schematic representation of the induction of ferroptosis by FHA NPs as an anticancer therapeutic. Schematic illustration of the strategy for the construction of FHA NPs and the sequential events of FHA NPs mediated by iron and HA: tumor-specific binding, cellular internalization, FHA NPs degradation and iron release, ROS generation, and lipid peroxidation.

Fig. 2

Characterization of FHA NPs. (A) Comparison of the UV–visible spectrum of HA, FeCl₂, FeCl₃, and different concentrations of FHA NPs. The absorbance spectra were measured using a UV–visible spectrophotometer. (B) X-ray diffraction patterns of (C) SEM images of FHA NPs. (D) TEM images of FHA NPs. (E) Quantification of iron (Fe) in internalized NPs measured using ICP-OES. (F) The absorption spectrum of FHA NPs, FHA NPs treated with 1,10-phenanthroline, and FHA NPs treated with hydroxylamine hydrochloride and 1,10-phenanthroline. (G) Relative comparison and photograph of hemolytic activity according to different concentrations of FHA NPs. (H) Stability of FHA NPs based on the size and polydispersity index (PDI) over a six-month period. The graph presents the change in particle size and PDI under storage conditions.

Fig. 3

Verification of viability and ROS generation in HFB, MCF7, HCT116, and A549 ​cells after treatment with FHA NPs. Viability in (A) HFB cells, (B) MCF7, (C) HCT116, and (D) A549 cancer cells after incubation with various concentration of FHA NPs for 1, 3, 6, and 12 ​h, separately, as determined using CCK-8 assays. (n ​= ​5) The value is indicated as mean ​± ​SD. (E) Flow cytometry analysis for CD44 expression in HFB, MCF7, HCT116, and A549 ​cells. Line, isotype control; color, CD44 expression. (F) Live/Dead assay in HFB, MCF7, HCT116, and A549 ​cells incubated with FHA NPs for 12 ​h as visualized with fluorescence microscopy. The green color and red color indicate living and dead cells, respectively. (scale bar: 100 ​μm) ​(G) Fluorescent images of CellROX Orange, representing ROS production. Blue and red indicate nuclear and ROS expression, respectively. (scale bar: 50 ​μm) ​(H) Flow cytometric analysis of intracellular ROS induction in cancer cell lines. Flow cytometric analysis was performed 12 ​h after treatment with various concentrations of FHA NPs.

Fig. 4

Qualitative and quantitative confirmation of intracellular Fe content. (A) Microscopic images of MCF7, HCT116, and A549 ​cells incubated with different concentrations of FHA NP and stained with Prussian blue and red counterstain. The cytoplasm is light pink, and the nucleus is dark pink. Iron particles are depicted as blue dots. (scale bar: 20 ​μm) 3D tomography image of A549 ​cells (B)without and (C)with FHA NPs treatment confirmed through refractive index (RI) values. Yellow and red dots represent refractive index (RI) values of the cell and FHA NPs, and blue represents DAPI staining, respectively. (scale bar: 5 ​μm) Quantification of internalized FHA NPs in (D) HFB, (E) MCF7, (F) HCT116, and (G) A549 ​cells based on ICP-AES and uptake ratio concentration.

Fig. 5

Real-time RT-PCR analysis and verification of the inhibitory effect. (A) Schematic diagram of cell death mechanism of ferroptosis through downregulation of FHA NP. Real-time RT-PCR analysis of (B) BAX, (C) RIPK1, and (D) GPX4 mRNA expression in MCF7, HCT116, and A549 ​cells. The internal control was used as β-Actin mRNA. Each bar represents mean ​± ​SD. (∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001) (E) Western blot analysis of β-Actin, BAX, RIPK1, and GPX4 expression in MCF7, HCT116, and A549 ​cells. Confirmation of inhibition effect of Ferrostatin-1, α-tocopherol (vitamin E), and RSL3 in (F) HFBs as well as (G) MCF7, (H) HCT116, and (I) A549 ​cell lines. Each cell line was treated with RSL3 in the presence or absence of ferostatin-1 and vitamin E, and cell viability was confirmed. Each was performed with three independent samples and values were represented as mean ​± ​SD.

Fig. 6

Confirmation of ROS and Lipid peroxidation (LPO) expression levels according to the presence of Ferrostatin-1 and lysosome and mitochondrial changes after treated FHA NPs. (A) Fluorescence images to confirm ROS generation in the presence or absence of Ferrostatin-1(Fer-1) under exposure to FHA NPs in MCF7, HCT116, and A549 ​cells. (scale bar: 50 ​μm) ​(B) FACS analysis to confirm ROS generation in the presence or absence of Fer-1 exposed to FHA NPs in MCF7, HCT116, and A549 ​cells. (C) Fluorescence images to confirm LPO levels in the presence or absence of Fer-1 under exposure to FHA NPs in MCF7, HCT116, and A549 ​cells. (scale bar: 50 ​μm) (D) FACS analysis to confirm LPO levels in the presence or absence of Fer-1 exposed to FHA NPs in MCF7, HCT116, and A549 ​cells. (E) Fluorescent images of MitoTracker staining to assess the mitochondrial morphology in A549 ​cells. Blue, red, and green represent nuclear, mitochondria, and actin filaments, respectively. (scale bar: 20 ​μm) ​(F) Fluorescent images of MitoTracker staining in A549 ​cells. Blue, red, and green represent nuclear, lysosome, and actin filaments, respectively. (scale bar: 20 ​μm) ​(G) FerroOrange and LysoTracker staining in A549 ​cells. Blue, green, and red represent nuclear, FerroOrange, and lysosome, respectively. (scale bar: 20 ​μm).

Fig. 7

FHA NP-mediated inhibition of tumor growth and progression in vivo. (A) Schematic diagram of the treatment used in vivo experiments. (B) The tumor volume differences of A549 tumor-bearing mice administered PBS and FHA NPs for 21 days. Quantitative analysis of tumor weight of (C) PBS groups and (D) FHA NPs groups for 21 days. (E) Photograph of the excised tumor at indicated time points for day 21. (F) MRI imaging in PBS- and FHA NP-injected groups to confirm Fe accumulation in organs and tumors. (G) Histological analysis of tumor sections stained with H & E. (scale bar: 50 ​μm) ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001.