H₂O₂-replenishable and GSH-depletive ROS ‘bomb’ for self-enhanced chemodynamic therapy

Abstract

Chemodynamic therapy (CDT) is an emerging strategy of tumor therapy that utilizes the Fenton reagent to kill tumor cells by disproportionation of H₂O₂ into hydroxyl radical (˙OH). However, insufficient endogenous H₂O₂ confines the antitumor efficacy of CDT. Additionally, the overexpressed glutathione (GSH) exhibits a potent scavenging effect on cytotoxic ˙OH, which further diminishes the efficacy of CDT. Though tremendous efforts have been done, engineering CDT agents with efficient and specific H₂O₂ self-supplying and GSH-depletion is promising but remains a great challenge. Herein, Fe³⁺–chelated CaO₂ nanoparticles (CaO₂–Fe NPs) are constructed as ROS ‘bomb’. In the tumor microenvironment, CaO₂–Fe NPs can release Fe²⁺ by the reduction of GSH, and the remaining CaO₂ reacts with H⁺ to selectively generate H₂O₂. The generated H₂O₂ can produce ˙OH under the catalysis of Fe²⁺ through the Fenton reaction, and re-oxidation from Fe²⁺ to Fe³⁺ endowing a long-lasting GSH-depletion, resulting in an improved CDT. These CaO₂–Fe NPs supply H₂O₂ and exhaust GSH simultaneously to achieve a self-enhanced CDT, and paves an emerging strategy to enhance the therapeutic efficacy of CDT by combining H₂O₂-replenishable and GSH-depletive together and realizing a self-enhanced Fenton reaction cycle.

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Introduction

Reactive oxygen species (ROS) based tumor therapy is an emerging therapeutic strategy to effectively induce tumor-cell apoptosis.^1–3 Among various ROS, cytotoxic hydroxyl radical (˙OH), which could be generated by H₂O₂via the Fenton reaction in an acidic environment is the most destructive and commonly applied type, which is defined as chemodynamic therapy (CDT).^4–7 Benefiting from the overproduction of H₂O₂ in tumors (100 μM–1 mM) than normal tissues, and the mild acidity of the tumor microenvironment (TME), CDT is regarded as a promising method for selective-tumor therapy with the help of catalysis by ferrous ions (Fe²⁺), manganese ions (Mn²⁺), or cuprous ions (Cu⁺).^8–11 However, some works recently suggested that the endogenous H₂O₂ in the tumor site is still insufficient to support effective CDT, which restricts the clinical application.^12–15 Therefore, the introduction of the H₂O₂-replenishable agents should be taken into consideration.

A few signs of progress have been made to increase the intratumoral H₂O₂ concentration by applying natural bio-enzymes such as glucose oxidase (GOx), nicotinamide adenine dinucleotide phosphate oxidase (NOX), and superoxide dismutase (SOD).^16–18 Nevertheless, these natural enzyme-based H₂O₂ supplements suffer from some potential issues, such as instability of biological activity, high cost, and reliance on the exogenous H₂O₂-precursors such as glucose, superoxide anion (O₂˙⁻) or O₂.^19,20 Encouragingly, it has been reported that inexpensive calcium peroxide (CaO₂) can steadily liberate a substantial amount of H₂O₂ under acidic conditions due to the presence of peroxy bond (–O–O–), and the production of H₂O₂ based on CaO₂ is independent of additional precursors.^21–25 Thus, based on the acidic environment of the tumor, CaO₂ is a practicable H₂O₂-replenishable ‘bomb’ for CDT to efficiently accumulate H₂O₂ with tumor specificity.

Conceivably, if ˙OH generated is eliminated by ROS scavengers such as glutathione (GSH), it is worth nothing. For this reason, efficient ROS generation in the tumor runs into the bottleneck due to elevated GSH level intratumorally (up to 10 mM) compared with that in normal tissue.^14,26–28 This problem could be potentially solved by employing some transition metal ions with variable valences, such as ions of iron, copper, and manganese, as recyclable GSH consumers. The transformation from a high-valence ion to its low-valence form reduces the concentration of GSH, and the resulting low-valence ion is a good catalyst for the Fenton reaction.^29–31 Noteworthy that during the Fenton reaction, the low-valence ion is re-oxidized to its high-valence form, giving a sustained depletion of GSH and release of ˙OH in GSH and H₂O₂-rich area.

Herein, a tumor-selective self-enhanced CDT ‘bomb’ is designed by using ferric ions (Fe³⁺)–chelated CaO₂ nanoparticles (CaO₂–Fe NPs). As elucidated in Fig. 1, under the tumor microenvironment, CaO₂ NPs reacted with specific H⁺ to form H₂O₂in situ as ROS ‘bomb’, and the chelated Fe³⁺ is reduced to Fe²⁺ by high-leveled GSH as the trigger. Subsequently, the Fenton reaction is activated by the generated H₂O₂ and Fe²⁺. Finally, ˙OH is produced for tumor therapy. Furthermore, the consumption of GSH enhances the CDT efficiency, and the re-oxidation of Fe²⁺ to Fe³⁺ endowing a long-lasting GSH-depletion. As a result, CaO₂–Fe NPs are able to selectively generate amounts of ROS to induce apoptosis of tumor cells with low systemic toxicity both in vitro and in vivo. These CaO₂–Fe NPs are good candidates for constructing ROS ‘bomb’ with endogenous replenishment of H₂O₂ and depletion of GSH, providing a novel strategy for improving tumor-selective CDT.

Fig. 1 The schematic illustration of CaO₂–Fe NPs as H₂O₂-replenishable and GSH-depletive ROS ‘bomb’ for self-enhanced CDT of the tumor.

Results and discussion

To obtain CaO₂–Fe NPs, CaO₂ NPs were first synthesized via a modified dopamine-assisted method, followed by mixing ferrous chloride to load iron ions.^21,32,33 The chelated iron contents were adjusted by different feeding ratios between CaO₂ and ferrous chloride (mass ratio). As shown in Table S1 (ESI†), contents of the chelated iron within CaO₂-Fe NPs were gradually improved with the increase of the feeding ratio. However, CaO₂–Fe NPs would not be formed when the feeding ratios were higher than 2 : 1. Thus, feeding ratios (4 : 1) were chosen to synthesize CaO₂–Fe NPs. The morphology of CaO₂–Fe NPs remained unchanged compared with the original CaO₂ NPs, and their diameters increased from 91 nm to 122 nm (Fig. 2a and Fig. S1, S2, ESI†). Subsequently, as confirmed using X-ray diffraction (XRD), the introduction of iron ions could not influence the phase of CaO₂ NPs (Fig. 2b). Strong and homogeneous iron signals were then observed from energy-dispersive X-ray spectroscopy (EDS) and EDS mapping, demonstrating the efficient binding of iron ions within CaO₂ NPs (Fig. 2c and Fig. S3, ESI†). To explore the valence state of chelated iron ions, X-ray photoelectron spectrometry (XPS) was applied. The central peak at ∼710.0 eV (Fe 2p_3/2) and the shakeup satellite peak at ∼724.0 eV (Fe 2p_1/2) demonstrated that Fe²⁺ was transformed into Fe³⁺, which might be oxidized by CaO₂ (Fig. 2d and e). The photoelectron peak at 532.5 eV of O 1s could be assigned to O–O, indicating the presence of peroxo groups (Fig. 2f).²⁴

Fig. 2 (a) TEM image of CaO₂–Fe nanoparticles (inset: an image at a higher magnification of CaO₂–Fe NPs). (b) XRD pattern of CaO₂ and CaO₂–Fe NPs. (c) EDS spectrum of CaO₂–Fe NPs. (d) Survey XPS spectra of CaO₂–Fe NPs. (e) High-resolution Fe 2p XPS spectra of CaO₂–Fe NPs. (f) High-resolution O 1s XPS spectra of CaO₂–Fe NPs.

Considering the significance of H₂O₂ for CDT, the H₂O₂ generation ability of CaO₂–Fe NPs was investigated using potassium permanganate (KMnO₄) as the indicator. As shown in Fig. 3a, the color of permanganate (MnO₄⁻) disappeared after adding CaO₂–Fe NPs to the acidic solution, suggesting the reduction of MnO₄⁻ to colorless Mn²⁺ by the generated H₂O₂. The dissociation of CaO₂–Fe NPs in acidic solution further verified the acid-activated H₂O₂ generation (Fig. S4, ESI†). In comparison, less H₂O₂ was generated from CaO₂–Fe NPs in a neutral environment (Fig. S5, ESI†). CaO₂–Fe NPs could maintain long-term stability in a neutral environment (Fig. S6, ESI†). It indicated that CaO₂–Fe NPs were good candidates for H₂O₂-replenishment in the acidic environment. These H₂O₂ suppliers could further release ˙OH induced by the Fenton reaction.

Fig. 3 (a) UV-Vis absorption spectra and photo (inset) of KMnO₄ after treating with H₂O₂, CaO₂ NPs, and CaO₂–Fe NPs in an acidic environment. (b) UV-vis absorption spectra and photo (inset) of MB after degradation by CaO₂–Fe NPs treated with different amounts of GSH at pH 5.4. (c) ESR spectra of CaO₂–Fe NPs treated with different amounts of GSH at pH 5.4 (5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap).

To evaluate the ROS triggered by CaO₂–Fe NPs, methylene blue (MB) was selected as the indicator. As can be seen in Fig. 3b and Fig. S7, S8 (ESI†), GSH is essential for ROS generation based on CaO₂–Fe NPs, due to the generation of Fenton-catalytic Fe²⁺ by the reduction of GSH. It is noteworthy that a high level of GSH was adverse for ROS generation in most reported cases due to the strong scavenging effect of GSH on ROS.^14,26 While CaO₂–Fe NPs exhibited an excellent ROS releasing capacity even when the concentration of GSH was at 10 mM, with the MB degradation efficiency appeared to be 99%. This phenomenon could be ascribed to the continuous depletion of GSH under the Fenton reaction cycle based on CaO₂–Fe NPs (Fig. S9, ESI†). During the GSH depletion and Fenton reaction cycle, Fe³⁺ was indispensable. In comparison with bare CaO₂ NPs without Fe³⁺ chelated, CaO₂–Fe NPs showed enhanced degradation of MB (Fig. S10, ESI†). Moreover, the ROS generation ability of CaO₂–Fe NPs was increased with the improvement of the chelated iron content (Fig. S11, ESI†). In addition, CaO₂–Fe NPs also showed a pH-dependent ROS due to the reliance on the generation of acidity of H₂O₂. These CaO₂–Fe NPs caused an apparent color degradation of MB under acidic conditions (pH 5.4) with the the assistance of GSH, but no significant change was observed under neutral conditions (pH 7.4) (Fig. S12, ESI†). The type of ROS produced by CaO₂–Fe NPs was further verified by the electron paramagnetic resonance (EPR) spin-trapping method. As shown in Fig. 3c, a characteristic 1 : 2 : 2 : 1 signal was obtained, indicating that the produced ROS by CaO₂–Fe NPs was ˙OH. These results suggested that CaO₂–Fe NPs were promising candidates for pH/GSH dual stimuli-activated CDT agents by H₂O₂ self-supplying and GSH-depletion.

Encouraged by the efficient production of ˙OH via CaO₂–Fe NPs with the assistance of GSH and H⁺, in vitro ˙OH generation was investigated due to the higher intracellular GSH concentration and lower pH value in tumor cells. By employing 2,7-dichlorofluorescin diacetate (DCFH-DA) as the ˙OH indicator, fluorescence imaging was carried out on 4T1 cells, which showed that the fluorescence signal of CaO₂–Fe NPs was dosage-dependent (Fig. S13, ESI†). Compared with CaO₂ NPs and FeCl₃ at the same dosage, CaO₂–Fe NPs exhibited significantly stronger green fluorescence, indicating the self-enhanced ˙OH was generated from CaO₂–Fe NPs in tumor cells (Fig. 4a and Fig. S14, ESI†). Considering the therapeutic effect of ˙OH, cell viability was then investigated by standard methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 4b, CaO₂–Fe NPs induced greater cell death by increasing concentrations, and the cytotoxic effect of the CaO₂–Fe NPs treated group was greater than that of FeCl₃ and CaO₂ NPs at the same concentration. Results from live/dead cell staining assay further confirmed these results, which revealed that only a small number of 4T1 cells remained viable after treatment with CaO₂–Fe NPs, while only a few cells were dead after treatment with FeCl₃ and CaO₂ NPs for 24 h (Fig. 4c and Fig. S15, ESI†).

Fig. 4 (a) Fluorescence images of DCFH-DA stained 4T1 cells after exposure to FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs for 4 h. The scale bar represents 100 μm. (b) Viability of 4T1 cells after 24 h of incubation with FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs. (n = 6, mean ± s.d., ***p < 0.001) (c) Fluorescence images of Calcein AM (green, live cells) and PI (red, dead cells) costained 4T1 cells after incubation with FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs for 24 h. The scale bar represents 100 μm. (d) Viability of L929 cells after 24 h of incubation with CaO₂–Fe NPs. (n = 6, mean ± s.d.) (e) Viability of 4T1 cells after 24 h of incubation with CaO₂–Fe NPs plus or without L-BSO. (n = 6, mean ± s.d., ***p < 0.001) (f) Intracellular GSH levels of 4T1 cells and L929 cells. (n = 3, mean ± s.d., ***p < 0.001) (g) Flow cytometry analysis of 4T1 cells treated with FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs for 24 h. (h) Viability of 4T1 cells after 24 h of incubation with CaO₂–Fe NPs plus or without NAC. (n = 6, mean ± s.d., ***p < 0.001).

Interestingly, the cell inhibition effect from CaO₂–Fe NPs was tumor cell-selective toxicity, which presented relatively low cytotoxicity toward normal cells (Fig. 4d and Fig. S16, ESI†). This phenomenon could be ascribed to the reliance of ˙OH generation on GSH concentration (Fig. 4e and Fig. S17, ESI†). After treating with CaO₂–Fe NPs, the intracellular GSH level was decreased, and the cell viability was reversed by down-regulating GSH by using L-buthionine sulfoximine (L-BSO) as the GSH inhibitor. In addition, GSH concentration within normal cells (L929) was much lower than cancerous cells (4T1) (Fig. 4f). As a result, reduced GSH could hardly trigger the generation of enough ˙OH on normal cells (Fig. S18, ESI†), and finally suppressed the side effect.

Flow cytometry was further used to investigate the type of cell death using the annexin V-FITC/PI detection kit. As shown in Fig. 4g, cell death induced by all groups was apoptosis, and the CaO₂–Fe NPs treated group had higher ratios of apoptotic cells (27%) than other groups. As ROS-mediated cell killing is regarded as the major pathway for apoptosis, the influence of N-acetyl-cysteine (NAC), a kind of ROS scavenger, on cell viability was then investigated. With the addition of NAC, cell apoptosis induced by CaO₂–Fe NPs was obviously reversed, indicating the cell inhibition was originating from the production of ROS in tumor cells (Fig. 4h). All these results suggested that CaO₂–Fe NPs was a GSH-enhanced CDT ‘bomb’ with self-supplied H₂O₂ to induce tumor cell apoptosis efficiently and selectively by ˙OH.

Tumor growth inhibition experiment was next performed by intravenous (i.v.) administration, inspired by outstanding treatment outcome of CaO₂–Fe NPs in vitro. In vivo biodistribution of CaO₂–Fe NPs was initially evaluated by labelling NIR dye (IR-783). As shown in Fig. S19 (ESI†), the CaO₂–Fe NPs could be accumulated in tumor tissue via the EPR effect, indicating that CaO₂–Fe NPs could serve as the ROS ‘bomb’ for self-enhanced chemodynamic therapy. The half-life of CaO₂–Fe NPs was 1.17 ± 0.45 h (Fig. S20, ESI†). Mice treated with saline (as the control group), FeCl₃, and CaO₂ NPs showed rapid tumor growth, while the size of tumors in CaO₂–Fe NPs injected mice was substantially inhibited (Fig. 5a and b). The therapeutic efficacy was also evidenced by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, which revealed extensive tumor cell apoptosis after treatment with CaO₂–Fe NPs (Fig. 5c). The effective generation of toxic ˙OH under TME with acidic pH and overexpressed GSH from CaO₂–Fe NPs, leading to a remarkable tumor growth inhibition effect. Moreover, no apparent body weight changes were observed in mice injected with CaO₂–Fe NPs during the whole period (Fig. 5d), and there was no obvious histological alteration in the major organs (Fig. S21, ESI†). No obvious physiological damages were observed in CaO₂–Fe NPs treated group through blood biochemistry and hematology analysis (Fig. 5e and Fig. S22, ESI†). Therefore, it is feasible to use CaO₂–Fe NPs as a smart CDT agent for effective tumor therapy with low toxicity.

Fig. 5 (a) Relative tumor growth curves of 4T1 tumor-bearing mice after treatment with saline (control group), FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs. (n = 5, mean ± s.d., ***p < 0.001) (b) A representative photo of dissected tumors from the different groups on day 17 after administration. (c) Images of H&E and TUNEL stained sections of tumors from the different groups on day 17 after administration. The scale bar represents 100 μm. (d) Time-dependent body-weight curves of mice in different groups. (n = 5, mean ± s.d.) (e) Blood biochemistry analysis of healthy mice after intravenously injected with saline or CaO₂–Fe NPs for 17 days. (n = 3, mean ± s.d.).

Conclusions

In summary, an H₂O₂-replenishable and GSH-depletive ROS ‘bomb’ was successfully constructed for self-enhanced chemodynamic tumor therapy. After reaching tumor tissues, these ROS ‘bomb’, CaO₂–Fe NPs, could be triggered due to the generation of Fe²⁺ ions by GSH. Meanwhile, amounts of H₂O₂ were generated by the reaction between CaO₂ and H⁺. Eventually, with the accumulation of H₂O₂ as well as Fe²⁺ locally, a Fenton reaction cycle was achieved by continuously consuming GSH to output massive ROS, resulting in the improvement of the CDT efficacy by H₂O₂-supplementing and GSH-depletion. Both, in vitro and in vivo results demonstrated that CaO₂–Fe NP presented an inspiring antitumor performance as well as low systemic toxicity. Therefore, these CaO₂–Fe NPs could be regarded as a promising candidate for combining pH/GSH-responsive and GSH-depletion for CDT.

Experimental section

Materials

Calcium chloride (CaCl₂, 96%), ammonium hydroxide (NH₃·H₂O, 28%), dopamine (99%), ferrous chloride (FeCl₂, 98%), methylene blue (MB, 70%), N-acetyl cysteine (NAC), and glutathione (GSH, 99%) were obtained from J&K Scientific Ltd (Beijing, China). Ethanol (C₂H₆OH, 99.7%) and hydrogen peroxide (H₂O₂, 30%) were purchased from the Juhua Group Corporation.

Characterization

X-Ray diffraction patterns (XRD) was recorded using the X’Pert PRO X-ray diffractometer with Cu Kα (λ = 1.54 Å). Transmission electron microscopy (TEM) was performed using a FEI Tecnai G2 F30 microscope. X-Ray photoelectron spectroscopy (XPS) was performed using the Axis Ultra imaging photoelectron spectrometer (Kratos Analytical Ltd). Dynamic light scattering (DLS) measurements were conducted using the Zetasizer Nano ZS (Malvern). The concentrations of Fe and Ca were quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES, NexION 350, PerkinElmer).

Synthesis of CaO₂ NPs and CaO₂–Fe NPs

CaCl₂ (0.1 g) and dopamine (0.003 g) were first dissolved in ethanol (15 mL) with the help of ultrasound. Subsequently, NH₄OH (1 mL) was added under magnetic stirring. Afterwards, H₂O₂ solution (0.2 mL) was injected slowly. The product (CaO₂ NPs) was finally collected by centrifugation (8000 rpm), washed with ethanol three times, and redispersed in ethanol. CaO₂ NPs were then reacted with FeCl₂ to form CaO₂–Fe NPs.

Colorimetric assay of peroxo groups

An aqueous solution containing KMnO₄ (50 μg mL⁻¹) and HCl (0.1 M) was first prepared. Subsequently, a certain amount of CaO₂ NPs, CaO₂–Fe NPs, or H₂O₂ was added into the mixture for 5 min. Finally, the mixture was measured by UV-vis spectra.

Chemodynamic activity of CaO₂–Fe NPs

The degradation of methylene blue (MB) was used for quantitative analysis of ROS production based on CaO₂–Fe NPs. In particular, the absorbance at λ = 644 nm of MB solution (25 mg L⁻¹) in pH 7.4 or pH 5.4 with or without different concentrations of GSH (0, 1, 2, 3, 4, 5, and 10 mM) was measured before and after adding of 20 μg CaO₂ NPs or CaO₂–Fe NPs for 3 hours.

To confirm the ROS type from CaO₂–Fe NPs, ESR spectroscopy was used. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was utilized as the spin trap. DMPO solution (40 μL, 100 mmol L⁻¹) was added into CaO₂–Fe NP solution at different concentrations of GSH (0, 1, 3, and 10 mM) at pH 5.4. Subsequently, the above mixture (20 μL) was injected into a capillary, the results were recorded using a Bruker A300.

Cell culture

4T1 and L929 cell lines were obtained from Zhejiang Provincial People's Hospital. All biological reagents were purchased from Biological Industries. DMEM or 1640 with 10% FBS and 1% penicillin/streptomycin were treated as the cell culture medium. All cells were cultured in a cell incubator at 37 °C, 5% CO₂ and 100% humidity.

Cytotoxicity assays

MTT assay was tested to evaluate the in vitro cytotoxicity. First, 5 × 10³ per well 4T1 and L929 cells were seeded into 96-well plates and incubated overnight. Subsequently, various amounts of FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs at the same Ca or Fe concentrations were added. After further incubation for 24 h, a fresh cell culture medium with 5% 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (MTT) was used to replace the culture medium with nanoparticles. Finally, dimethyl sulfoxide (DMSO, 100 μL) was used to replace the MTT solution and co-incubation for 4 h. Cell viability was measured using a Tecan m200. Furthermore, after 4T1 cells were treated after the above conditions, Calcein-AM and propidium iodide (PI) live/dead cell staining was used to further verify the cytotoxicity of CaO₂–Fe NPs.

Intracellular ROS levels detection

5 × 10⁴ per well 4T1 and L929 cells were plated into a 24-well plate and incubated overnight. Then, cells were incubated with FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs at the same Ca or Fe concentrations for 4 h. After washing with PBS, cells were stained with DCFH-DA (10 μM) for 30 min. Later, PBS was used to remove the free DCFH-DA. Finally, the fluorescence images were obtained using a Nikon ECLIPSE Ti.

Assessment of apoptosis

Annexin-V/PI assay kit (Sony) was used to determine the apoptosis of 4T1 cells treated with CaO₂–Fe NPs using flow cytometry. In particular, 2 × 10⁵ per well 4T1 cells were plated into a 6-well plate and incubated for 12 h. Afterwards, cells were treated with FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs at the same Ca or Fe concentrations for a further 24 h. After washing with PBS, cells were detached by trypsin. Finally, apoptosis was detected using flow cytometry (ACEA NovoCyte) using PI vs. Annexin V plots.

Animal modal

All animal experiments were performed abiding by the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Zhejiang Provincial People's Hospital, Hangzhou, China. 6-Week female Balb/c mice were provided by Shanghai Sippe-Bk Lab Animal Co., Ltd, Shanghai, China. 1 × 10⁶ 4T1 cells solution (0.1 mL cells in PBS) was subcutaneously injected into the right axillary of all mice to construct the tumor model.

Tumor inhibition and in vivo toxicity assay

After tumor volume reached 100 mm³, 20 mice were randomly divided into 4 groups (n = 5): mice were intravenously injected with saline (as the control group), FeCl₃, CaO₂ NPs, and CaO₂–Fe NPs at the same Ca or Fe concentrations. The whole experiment period was 15 days. All treatments were performed every three days and tumor volume and body weight were recorded 1-day after injection. Tumor volume was calculated using the formula as 0.5 × (length × width²).

On the 17th day, all mice were executed. Subsequently, tumors and major organs (heart, liver, spleen, lung, and kidney) were removed and stored in formalin. After the section of the tumors and major organs, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were then detected. A Nikon ECLIPSE Ni-U was used to observe the slides.

Statistical analysis

The test data are shown as mean ± s.d. The student's two-tailed t-test was used to calculate the statistical comparisons. * means p < 0.05, ** means p < 0.01, *** means p < 0.001. p < 0.05 was regarded as statistically significant.

Author contributions

Fan Zhao: carried out all experiments and performed the statistical analysis, contributed to discussion, writing – original draft. Jiayu Yao: participated in animal studies and molecular biology experiments. Yu Tong: participated in animal studies and molecular biology experiments. Qing Xu: participated in molecular biology experiments. Dan Su: participated in molecular biology experiments. Juan Li: supervision. Yao Ying: supervision. Wangchang Li: supervision. Liang Qiao: supervision. Jingwu Zheng: supervision. Wei Cai: supervision. Xiaozhou Mou: conceptualization, supervision, writing – reviewing and editing. Shenglei Che: conceptualization, supervision, writing – reviewing and editing. Jing Yu: carried out all experiments and performed the statistical analysis, conceptualization, writing – review & editing. Yanglong Hou: conceptualization, supervision, writing – reviewing and editing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (52073258 and 81672430), Natural Science Foundation of Zhejiang Province (LY20E020017 and LQ19H160016), Young Elite Scientist Sponsorship Program by CAST (2017QNRC001), the Fundamental Research Funds for the Provincial Universities of Zhejiang (RF-A2019004), and Foundation of Health Department of Zhejiang Province (2018KY239).

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Footnote

† Electronic supplementary information (ESI) available: Supplementary figures and discussions. See DOI: 10.1039/d1ma00646k

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