Cooperation of endogenous and exogenous reactive oxygen species induced by zinc peroxide nanoparticles to enhance oxidative stress-based cancer therapy

Abstract

Reactive oxygen species (ROS)-generating anticancer agents can act through two different mechanisms: (i) elevation of endogenous ROS production in mitochondria, or (ii) formation/delivery of exogenous ROS within cells. However, there is a lack of research on the development of ROS-generating nanosystems that combine endogenous and exogenous ROS to enhance oxidative stress-mediated cancer cell death. Methods: A ROS-generating agent based on polymer-modified zinc peroxide nanoparticles (ZnO₂ NPs) was presented, which simultaneously delivered exogenous H₂O₂ and Zn²⁺ capable of amplifying endogenous ROS production for synergistic cancer therapy. Results: After internalization into tumor cells, ZnO₂ NPs underwent decomposition in response to mild acidic pH, resulting in controlled release of H₂O₂ and Zn²⁺. Intriguingly, Zn²⁺ could increase the production of mitochondrial O₂·^- and H₂O₂ by inhibiting the electron transport chain, and thus exerted anticancer effect in a synergistic manner with the exogenously released H₂O₂ to promote cancer cell killing. Furthermore, ZnO₂ NPs were doped with manganese via cation exchange, making them an activatable magnetic resonance imaging contrast agent. Conclusion: This study establishes a ZnO₂-based theranostic nanoplatform which achieves enhanced oxidative damage to cancer cells by a two-pronged approach of combining endogenous and exogenous ROS.

Keywords: cancer therapy; magnetic resonance imaging; pH-responsiveness; reactive oxygen species; zinc peroxide nanoparticles.

Figure 1

Schematic illustration of theranostic ZnO₂ NPs for MRI and enhanced oxidative stress-based cancer therapy. Upon endocytosis by tumor cells, ZnO₂ NPs undergo dissociation in response to mild acidic pH, causing the release of H₂O₂ and Zn²⁺. The exogenously released H₂O₂ and Zn²⁺, that can increase the production of mitochondrial O₂·^- and H₂O₂ by inhibiting the electron transport chain (ETC), act synergistically to promote cancer cell killing through the cooperation of endogenous and exogenous ROS. Moreover, Mn-doping via partial cation exchange imparts ZnO₂ NPs with pH-activated MRI contrast ability.

Figure 2

(A) TEM image and (B) DLS data of ZnO₂ NPs. (C) Raman spectra of ZnO NPs and ZnO₂ NPs. (D) XPS survey spectra and O 1s peak (inset) of ZnO₂ NPs.

Figure 3

Release profiles of (A) Zn²⁺ and (B) H₂O₂ from ZnO₂ NPs at different pH values. (C) TEM images of ZnO₂ NPs after 2 h of incubation in pH 7.4 (left) and pH 5.5 (right) buffer solutions. (D) Fluorescence images of zinquin ethyl ester-stained U87MG cells after incubation with different concentration of ZnO₂ NPs for 4 h. Scale bar, 50 μm.

Figure 4

(A) DCF fluorescence of U87MG cells after different treatments. [H₂O₂] = 200 μM, [ZnCl₂] = 200 μM. Scale bar, 50 μm. (B) Cell viability after 24 h of exposure to H₂O₂, ZnCl₂, or H₂O₂ plus ZnCl₂ (molar ratio, 1:1).

Figure 5

(A) In vitro anticancer activity of ZnO₂ NPs after 24 h of incubation. (B) DCF fluorescence of U87MG cells after 4 h of incubation with 10 μg mL^-1 ZnO₂ NPs. Scale bar, 50 μm. (C) Calcein-AM (green, live cells) and PI (red, dead cells) co-stained fluorescence images of cells treated with different concentrations of ZnO₂ NPs for 24 h. Scale bar, 100 μm. (D) Flow cytometry data showing apoptosis in U87MG cells after exposure to ZnO₂ NPs for 12 h.

Figure 6

(A) Scheme showing the preparation of Mn-doped ZnO₂ NPs through a facile cation-exchange approach. (B) EDS mapping of Mn-ZnO₂ NPs. (C) The r₁ values of Mn-ZnO₂ NPs under different pH conditions. (D) T₁-weighted MRI of U87MG tumor-bearing mice after intravenous injection of Mn-doped ZnO₂ NPs. The red circles indicate the tumor area.

Figure 7

(A) Tumor growth curves of U87-bearing mice injected intravenously with different formulations. (B) Survival curves of mice in different groups. (C) Body weight changes of mice during the observation period. (D) TUNEL and (E) H&E staining of tumor tissues derived from different groups. Scale bar in d, 50 μm.