Cu-Ferrocene-Functionalized CaO2 Nanoparticles to Enable Tumor-Specific Synergistic Therapy with GSH Depletion and Calcium Overload

Abstract

The conversion of endogenous H₂ O₂ into toxic hydroxyl radical (^• OH) via catalytic nanoparticles is explored for tumor therapy and received considerable success. The intrinsic characteristics of microenvironment in tumor cells, such as limited H₂ O₂ and overexpressed glutathione (GSH), hinder the intracellular ^• OH accumulation and thus weaken therapeutic efficacy considerably. In this study, fine CaO₂ nanoparticles with Cu-ferrocene molecules at the surface (CaO₂ /Cu-ferrocene) are successfully designed and synthesized. Under an acidic condition, the particles release Ca²⁺ ions and H₂ O₂ in a rapid fashion, while they can remain stable in neutral. In addition, agitated production of ^• OH occurs following the Fenton reaction of H₂ O₂ and ferrocene molecules, and GSH is consumed by Cu²⁺ ions to avoid the potential ^• OH consumption. More interestingly, in addition to the exogenous Ca²⁺ released by the particles, the enhanced ^• OH production facilitates intracellular calcium accumulation by regulating Ca²⁺ channels and pumps of tumor cells. It turns out that promoted ^• OH induction and intracellular calcium overload enable significant in vitro and in vivo antitumor phenomena.

Keywords: CaO2; Cu-ferrocene; GSH depletion; calcium overload; synergistic tumor therapy.

Figure 1

Schematic illustration of the synthesis and functioning procedures of CaO₂/Cu–ferrocene.

Figure 2

Characteristics of CaO₂/Cu–ferrocene. a) Scanning electron microscopy (SEM), b) transmission electron microscopy (TEM), c) element mapping images, and d) XRD pattern of CaO₂/Cu–ferrocene. e) Zeta potential and f) hydrodynamic dimension of CaO₂ and CaO₂/Cu–ferrocene particles. g) XPS spectrum of CaO₂/Cu–ferrocene. h) XPS high‐resolution spectrum of Cu 2p. i) XPS high‐resolution of Fe 2p.

Figure 3

a) Release profiles of H₂O₂ from CaO₂ and CaO₂/Cu–ferrocene under different pH. b) pH variation in acetate buffer solution with the addition of CaO₂ and CaO₂/Cu–ferrocene. c) GSH consumption of CaO₂/Cu–ferrocene with different concentrations. d) UV–vis absorbance spectra. e) Time‐course absorbance at 655 nm with different concentrations of CaO₂/Cu–ferrocene in TMB solution. f) ESR spectra of CaO₂/Cu–ferrocene at different pH values.

Figure 4

a) HL‐7702 and RAW 264.7 cell viabilities with different concentrations of CaO₂/Cu–ferrocene. 4T1 viabilities with different concentrations of CaO₂/Cu–ferrocene at varied b) pH and c) incubation time. d) Flow cytometry analysis and e) Live and Dead cell staining of 4T1 cells with different concentrations of CaO₂/Cu–ferrocene (***p < 0.001, **p < 0.01, or *p < 0.05).

Figure 5

a) Intracellular H₂O₂ in 4T1 cells cultured without or with CaO₂/Cu–ferrocene. b) Fluorescence images of 4T1 cells incubated with CaO₂/Cu–ferrocene and varied pH after DCFH‐DA staining for ROS detection. Fluorescence images of 4T1 cells incubated with different concentrations of CaO₂/Cu–ferrocene after staining for c) intracellular GSH depletion and d) intracellular Ca²⁺ ions’ accumulation. e) Expression of Calpain‐1, PMCA4, TRPA1, Caspase‐3, Bcl‐2, and BAX in 4T1 cells treated with different groups in inducing cellular apoptosis. f) Schematic diagram of functioning of CaO₂/Cu–ferrocene as a multifunctional platform for promoted ROS induction and calcium overload.

Figure 6

In vivo assay. a) Experimental procedures of injection treatment. b) Variation of body weight over 14 days. c) Tumor photographs and d) tumor weights from different mice groups after 14 days. e) Variation of tumor volume in different groups during 14 day treatment. f) Images of H&E‐ and ki67‐stained tumor slices from different groups after 14 days (***p < 0.001, **p < 0.01, or *p < 0.05).

Figure 7

a) Variation of tumor volume in Group 5 treated with intravenous injection of CCF. b) In vivo pharmacokinetic curves in 72 h. c) Distribution of nanoparticles in different organs at 24 and 72 h after injecting with CCF. d) H&E‐stained slices from different organs on days 0, 1, 3, and 14 (***p < 0.001, **p < 0.01, or *p < 0.05).