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

Abstract The conversion of endogenous H2O2 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 H2O2 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 H2O2 in a rapid fashion, while they can remain stable in neutral. In addition, agitated production of •OH occurs following the Fenton reaction of H2O2 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.

Characterization. Microstructure images of samples were obtained using transmission electron microscopy (TEM, HITACHI HT-7700) and field-emission scanning electron microscopy (FESEM, Phenom Pharos), respectively. Zeta potential and dynamic light scattering were measured by Zetasizer Nano-ZS (0.3-10000 nm). X-ray diffraction pattern was obtained by X-ray diffractometer with Cu Kα radiation (XRD, X'pert PRO MPD). The chemical composition and valence states of elements were measured by X-ray photoelectron spectroscopy (XPS, AXIS Supra). The UV-visible absorbance was examined by UV-vis spectrophotometer (Shimadzu, UV-2600). Hydroxyl radicals was detected by electron spin resonance spectroscopy (EPR, Bruker A300). Cell Counting Kit-8 (CCK-8), H 2 O 2 and GSH contents were recorded using a microplate reader. The fluorescence images of Live and Dead, intracellular reactive oxygen species (ROS) and Ca 2+ ions were obtained by an inverted fluorescent microscope (Nexcope, The USA).

Synthesis of CaO 2 and CaO 2 /Cu-ferrocene (CCF) nanoparticles. CaO 2 spherical aggregates
were synthesized following protocol the procedures reported previously [1] . CaCl 2 (0.1 g) and PVP (0.35 g) were dissolved in 15 mL ethanol, and 1 mL NH 3   Subsequently, the morphology of nanoparticles was observed by TEM. GSH depletion. Different quantities of CCF were dispersed in 6 mL GSH solution (1.5 mM, pH=7) at 37 °C. 0.5 mL supernatant was collected from the solution after centrifugation at different time points of the reaction (0, 2, 4, 6 and 12 h). Subsequently, 2.5 mL PBS and 50 μL 5,5'-Dithiobis-(2-nitrobenzoic acid) (10 mM, DTNB was used as the indicator of GSH) were added into the supernatant. The total amount of GSH was determined quantitatively according to the UV-vis absorbance standard curve of GSH at 407 nm.
To exclude the consumption of GSH concentration by the hydrogen peroxide induced by the hydrolysis of CaO 2 , the examination was carried out using CaO 2 and Cu-ferrocene, respectively. The reaction time was set at 1 hour, and the experimental procedure was maintained the same as stated above.
•OH generation. •OH generation was detected by the chromogenic reaction of TMB. Briefly, TMB (8 mM) was added into 3 mL ABS (0.1 M, pH = 5) with CCF at different concentrations. The absorption spectra and kinetic curves were recorded by a UV-vis spectrometer. The generation of •OH was further identified by electron spin resonance (ESR) spectroscopy with 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, which can be recognized with a characteristic 1:2:2:1 hydroxyl radical signal.

In vitro study.
Cell viability assay. The cell viability of CCF was examined by CCK-8. Cell compatibility was evaluated by normal cell lines, HL-7702 (human liver cell) and RAW264.7 (mouse mononuclear macrophages). Briefly, cells (10,000 per well) were seeded into a 96-well plate for 12 h. The medium was removed, and fresh medium containing CCF (0-80 μg/mL) was added and incubated for further 24 h. 10 μL CCK-8 was added per well and incubated for 1 h.
The results were acquired using a microplate reader for absorbance at 450 nm.
In addition, the in vitro antitumor properties were examined by CCK-8 assay using 4T1 cells (mouse breast cancer cell). Briefly, 4T1 cells (10,000 per well) were seeded into a 96-well plate for 12 h. Then, the medium was removed and fresh medium containing CCF (0-80 μg/mL) was added for further 24 h. The results were acquired using a microplate reader at 450 nm. To simulate acidic tumor microenvironment, the pH of cell culture was set at 6.5 by the addition of HCl solution (1 M, 15 μL) into the medium (980 μL), and CCF was added subsequently.
The Live and Dead assay was also used to confirm the cytotoxicity of CCF to tumor cells.  [2] .
Fluo-4 AM is a cell-permeable Ca 2+ indicator, which reacts with the esterase within the cell to generate non-fluorescent Fluo-4. The presence of Ca 2+ ions endows Fluo-4 with strong fluorescence. To detect excessive Ca 2+ ions, Fluo-4 AM (10 μM) was applied in accordance with the same procedures above.
In order to assess the magnitude of calcium overload, the intracellular calcium content was examined by inductively coupled plasma optical emission spectrometer (ICP-OES).

Intracellular H 2 O 2 generation. Intracellular H 2 O 2 content was examined by a H 2 O 2 assay kit.
Briefly, 4T1 cells were seeded into 6-well plates and incubated at 37 °C for 12 h. After removal of the medium, the cells were incubated with different concentrations of CCF. After 8 h, cell lysis buffer was added into the well and the supernatant was collected by centrifugation. H 2 O 2 assay kit was then added, and the results were recorded by a microplate reader at 560 nm.

Intracellular GSH consumption.
The membrane-permeant naphthalene-2,3-dicarboxaldehyde (NDA), an extremely sensitive fluorescence probe, was used to determine intracellular glutathione content [3] . NDA can react with GSH to produce strong fluorescence isoindole adducts. The specific operation was similar to ROS detection, and the concentration of working solution was adjusted at 50 μM.
The GSH content was quantitatively detected using DTNB chromogenic method. After incubation with 40 μg/mL CCF for 8 h, cell lysis buffer was added into the well, and the supernatant was collected by centrifugation. Then DTNB was added and the results were recorded by a microplate reader at 405 nm. Statistical analysis. All data in this article are presented as mean ± standard deviations.
Student's t-tests were used to evaluate the comparison results between experimental groups.
Variations in the data were considered to be significant when ***p < 0.001, **p < 0.01 or *p < 0.05.