Superparamagnetic iron oxide nanoparticles: amplifying ROS stress to improve anticancer drug efficacy

Abstract

Superparamagnetic iron oxide nanoparticles (SPION) are an important and versatile nano- platform with broad biological applications. Despite extensive studies, the biological and pharmacological activities of SPION have not been exploited in therapeutic applications. Recently, β-lapachone (β-lap), a novel anticancer drug, has shown considerable cancer specificity by selectively increasing reactive oxygen species (ROS) stress in cancer cells. In this study, we report that pH-responsive SPION-micelles can synergize with β-lap for improved cancer therapy. These SPION-micelles selectively release iron ions inside cancer cells, which interact with hydrogen peroxide (H(2)O(2)) generated from β-lap in a tumor-specific, NQO1-dependent manner. Through Fenton reactions, these iron ions escalate the ROS stress in β-lap-exposed cancer cells, thereby greatly enhancing the therapeutic index of β-lap. More specifically, a 10-fold increase in ROS stress was detected in β-lap-exposed cells pretreated with SPION-micelles over those treated with β-lap alone, which also correlates with significantly increased cell death. Catalase treatment of cells or administration of an iron chelator can block the therapeutic synergy. Our data suggest that incorporation of SPION-micelles with ROS-generating drugs can potentially improve drug efficacy during cancer treatment, thereby provides a synergistic strategy to integrate imaging and therapeutic functions in the development of theranostic nanomedicine.

Keywords: Fenton reaction; reactive oxygen species; superparamagnetic iron oxide nanoparticles; theranostic nanomedicine.; β-lapachone.

Figure S1

(Scheme 1). Schematic illustration of intracellular distribution of pH-sensitive SPION-micelles, iron ion release, subsequent Fenton reactions with NQO1-dependent H₂O₂ generated from β-lapachone (β-lap) futile redox cycle to amplify ROS stress levels for improved antitumor efficacy. NQO1 - NAD(P)H: quinone oxidoreductase 1; SOD - superoxide dismutase.

Figure 1

(A) Transmission electron microscopy (TEM) image of a representative SPION-micelle sample (inset TEM image shows 5.8 ± 1.0 nm SPIO nanoparticles). (B) Release profiles of iron ions from SPION-micelles at various pH values. (C) Confocal laser scanning microscopy of intracellular distribution of rhodamine-labelled SPION-micelles in A549 NSCLC cells stained with LysoTracker green after 4 h incubation. Dissociation of SPION-micelles in acidic organelles resulted in activation of rhodamine signal (red), which co-localized with lysosomes (green).

Figure 2

(A) Fluorescent images of A549 cells treated with SPION-micelles and β-lap under different conditions using DHE staining for hydroxyl and superoxide radical detection. (B) Quantification of fluorescence intensity changes reflecting ROS levels in A549 NSCLC cells pretreated with or without SPION-micelles followed by exposure to β-lap. Fluorescent intensity of each image was quantified using ImageJ; values are means ± SE from at least 50 cells in triplicate experiments. (C) Western blot analyses of PAR formation in A549 cancer cells treated with different β-lap concentrations, with or without SPION-micelle pretreatment (0.07mM).

Figure 3

(A) Cell survival study of A549 cells with or without pretreatment of 0.14 mM SPION-micelles for 48 hrs followed by treatment with various β-lap concentrations. (B) Survival study of A549 cells pretreated with 0.14 mM SPION-micelles for 48 hrs, followed by 3 μM β-lap for 4 hrs with or without dicoumarol.

Figure 4

(A) Cytotoxicity of A549 NSCLC cells pretreated with SPION-micelles at 0.07 mM or 0.14 mM for 48 hrs followed by exposure to 3.0 μM β-lap for 4 hrs with or without the H₂O₂ scavenger, catalase (1000 U). (B) Survival study of A549 cells pretreated with 0.07 mM or 0.14 mM SPION-micelles for 48 hrs, with or without the iron chelator, deferoxamine (Def, 5 μg/mL), with or without 3 μM β-lap for 4 hrs. Data presented are means ± SE from experiments performed in triplicate.

Figure 5

Enhanced cell death mechanism at sublethal dose of β-lap as induced by SPION-micelles. Production of hydroxyl radicals are dramatically elevated in cancer cells exposed to β-lap and SPION-micelles, which results in DNA damage, Ca²⁺ release, and eventually PARP-1 hyperactivation. Catalase and deferoxamine can prevent the synergized lethality by consuming the H₂O₂ or chelating iron ions, respectively.