Zn_0.4Mg_0.6Fe₂O₄ nanoenzyme: a novel chemo-sensitizer for the chemotherapy treatment of oral squamous cell carcinoma

Abstract

Hypoxic and acidic environments are the two main components of the microenvironment contributing to the poor efficacy of chemotherapy drugs in the treatment of oral squamous cell carcinoma (OSCC). In this study, we synthesized a series of Zn_1–xMg_xFe₂O₄ nanomaterials with enzyme-like properties, including catalase (CAT)-like, peroxidase (POD)-like, and glutathione (GSH)-like activity in an acidic environment. Among them, Zn_0.4Mg_0.6Fe₂O₄ performed the best and effectively increased the efficacy of doxorubicin (DOX) chemotherapy for the treatment of OSCC with reduced cardiotoxicity. Therefore, Zn_0.4Mg_0.6Fe₂O₄ could serve as a novel chemosensitizer in the treatment of OSCC.

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1 Introduction

As one of the most malignant tumor-prone sites, the number of new cases and associated deaths due to malignant tumors in the oral cavity worldwide were 377 713 and 177 757 respectively in 2020, and the numbers are increasing every year.¹ Among these, oral squamous cell carcinoma (OSCC) accounts for 80–90%. Although radical resection is the preferred treatment, chemotherapy is still an important auxiliary method to treat or prolong life for patients with advanced/recurrence OSCC or patients who cannot tolerate surgery.² The armamentarium of available chemotherapy has been increasing rapidly in recent years, while the 5-year survival rate of advanced/recurrence OSCC remains less than 30%.^3,4 How to improve chemotherapy efficacy has become the key to prolonging the survival of patients with OSCC.

Hypoxic and acidic environments are two important characteristics of the tumor microenvironment. Oxygen is not only an important microenvironmental factor in the development of the organism and normal tissue homeostasis, but is also essential for oxidative metabolism, ATP production, and cell survival.⁵ In the process of tumor occurrence and development, the abnormal vascular system in the tumor microenvironment leads to obstacles in oxygen delivery, and at the same time, increased oxygen consumption due to tumor cell proliferation and immune cell infiltration eventually leads to hypoxia in the tumor microenvironment.⁶ Many studies have suggested that the hypoxic environment is closely related to tumor metastasis, tolerance to radiotherapy/chemotherapy, and a poor prognosis.^7,8 Moreover, the lack of oxygen in tumor tissues also leads to an increase in anaerobic glycolysis and to the production/secretion of H⁺ in cells. Eventually, the acidic metabolites accumulate due to the limited blood perfusion in the acidic environment in malignant tumor tissues. Studies have shown that an acidic environment could activate proteolytic enzyme activity, which is involved in tumor tissue remodeling and tumor invasion.⁹ Furthermore, the degree of acidity of tumor tissues has also been reported to be related to their degree of malignancy.¹⁰ Therefore, this feature may be exploited as a novel approach to overcome chemotherapy tolerance in patients with OSCC by designing drugs to correct the hypoxic state based on the acidic tumor environment.

Since Yan et al. reported the intrinsic peroxidase-like activity of ferromagnetic nanoparticles in 2007,¹¹ an increasing number of nanomaterials with enzyme-like characteristics (nanozymes) have been developed and applied in the biomedical field.¹² Compared to traditional natural enzymes, nanozymes are generally of low-cost and can be mass-produced. Additionally, previous studies have shown that catalase (CAT)/peroxidase (POD) mimic nanozymes such as MnFe₂O₄ could catalyze H₂O₂ oxygen production, which could correct the hypoxic state in tumor tissue and may produce reactive oxygen species (ROS) that could inhibit tumor cell proliferation.^13–15 This suggests that we may be able to correct the hypoxic state by constructing appropriate nanozymes that reduce tolerance to chemotherapy in OSCC patients.

In this paper, a series of Zn_1–xMg_xFe₂O₄ nanozymes were synthesized and, of these, Zn_0.4Mg_0.6Fe₂O₄ exhibited the highest catalytic efficiency of POD-like, CAT-like, and GSH-like activity. Furthermore, the properties of POD–CAT and glutathione (GSH) activity of Zn_0.4Mg_0.6Fe₂O₄ were verified in vitro. Finally, we verified whether Zn_0.4Mg_0.6Fe₂O₄ could reduce chemotherapy tolerance and improve the effect of chemotherapy on OSCC, and the results showed that Zn_0.4Mg_0.6Fe₂O₄ could serve as a promising chemosensitizer in OSCC management. The main scheme of our study is depicted below (Fig. 1).

Fig. 1 Diagram of the chemosensitization mechanism of the Zn_0.4Mg_0.6Fe₂O₄ nanoenzyme.

2 Results and discussion

2.1 Synthesis and characterization of Zn_1–xMg_xFe₂O₄

A series of Zn_1–xMg_xFe₂O₄ molecules were successfully synthesized as shown in Fig. 2A and the X-ray diffraction (XRD) spectra are given in Fig. 2B. Their hydrodynamic diameters measured by dynamic light scattering (DLS) were 21.96 ± 3.10 nm (MgFe₂O₄, polymer dispersity index (PDI): 0.02), 23.41 ± 5.04 nm (Zn_0.2Mg_0.8Fe₂O₄, PDI: 0.05), 28.52 ± 5.36 nm (Zn_0.4Mg_0.6Fe₂O₄, PDI: 0.04), 29.67 ± 6.24 nm (Zn_0.6Mg_0.4Fe₂O₄, PDI: 0.04), 40.80 ± 9.76 nm (Zn_0.8Mg_0.2Fe₂O₄, PDI: 0.06), and 48.70 ± 15.43 nm (ZnFe₂O₄, PDI: 0.10), as shown in Fig. 2C. The diameter of Zn_1–xMg_xFe₂O₄ increased with an increase in the Zn ratio. The zeta potentials of obtained Zn_1–xMg_xFe₂O₄ were −31.63 ± 1.31 mV (MgFe₂O₄), −36.5 ± 1.75 mV (Zn_0.2Mg_0.8Fe₂O₄), −29.83 ± 2.05 mV (Zn_0.4Mg_0.6Fe₂O₄), −27.93 ± 1.34 mV (Zn_0.6Mg_0.4Fe₂O₄), −30.63 ± 2.11 mV (Zn_0.8Mg_0.2Fe₂O₄), and −26.93 ± 1.57 mV (ZnFe₂O₄), as shown in Fig. 2D. The morphology was evaluated using a transmission electron microscope (TEM) and the selected area electron diffraction (SAED) showed that these nanoparticles had a morphology of homogeneously dispersed spheres and a single-crystal structure, as shown in Fig. 2E.

Fig. 2 Characterization of Zn_1–xMg_xFe₂O₄. (A) Camera images of Zn_1–xMg_xFe₂O₄, (B) XRD spectra of Zn_1–xMg_xFe₂O₄, (C) DLS of Zn_1–xMg_xFe₂O_4, (D) zeta potential of Zn_1–xMg_xFe₂O₄ and (E) TEM images of Zn_1–xMg_xFe₂O₄, and the insets show the SAED patterns, respectively.

2.2 CAT-like, POD-like, and GSH-like properties of Zn_1–xMg_xFe₂O₄

2.2.1 Zn_0.4Mg_0.6Fe₂O₄ exhibited excellent CAT-like activity. It is effective in reducing the tolerance to OSCC chemotherapy by increasing the content of O₂. As shown in Fig. 3A, H₂O₂ was abundant in OSCC tumor tissues compared to normal tissues, which could provide the substrate for the generation of O₂. Therefore, both the consumption of H₂O₂ and the generation of O₂ have been used to assess the CAT-like activity of Zn_1–xMg_xFe₂O₄. In terms of consumption of H₂O₂, Zn_0.4Mg_0.6Fe₂O₄, Zn_0.8Mg_0.2Fe₂O₄, and ZnFe₂O₄ showed CAT-like activity in an acid/neutral environment (pH = 5.8/7.4) compared to the control group, while Zn_0.6Mg_0.4Fe₂O₄, Zn_0.2Mg_0.8Fe₂O_4, and MgFe₂O₄ did not (Fig. 3B, C and S1†). The amount of H₂O₂ consumption at 3 h was used to explore the influence of the Zn/Mg ratio on the CAT-like catalytic efficiency of Zn_1–xMg_xFe₂O₄. As shown in Fig. 3D, Zn_0.4Mg_0.6Fe₂O₄ has decomposed the maximum amount of H₂O₂ than the other Zn_1–xMg_xFe₂O₄ nanozymes. Furthermore, as shown in Fig. 3E, Zn_0.4Mg_0.6Fe₂O₄ was also the O₂-producing Zn_1–xMg_xFe₂O₄. Therefore, Zn_0.4Mg_0.6Fe₂O₄ has potential application value in correcting the tumor hypoxic microenvironment of OSCC and improving the chemotherapy effect.

Fig. 3 Properties of the CAT-like activity of Zn_0.4Mg_0.6Fe₂O₄. (A) The H₂O₂ content in OSCC tumor tissues and normal tissues. (B) H₂O₂ consumption continues after treatment with Zn_1–xMg_xFe₂O₄ in an acidic environment. (C) Continuous consumption of H₂O₂ after treatment with Zn_0.4Mg_0.6Fe₂O₄ in different buffers (PBS, pH = 5.8 or 7.4). (D) Comparison of CAT-like activity at 3 h. (E) Continuous generation of O₂ after treatment with Zn_1–xMg_xFe₂O₄ in an acidic environment. ***: P < 0.001.

2.2.2 Zn_0.4Mg_0.6Fe₂O₄ exhibited excellent POD-like activity. ROS detection was performed to investigate the POD-like activity of Zn_1–xMg_xFe₂O₄. As shown in Fig. 4A, B and S2,† compared to the control group, all Zn_1–xMg_xFe₂O₄ exhibited POD-like activity in an acid environment (pH = 5.8), and no POD-like activity was observed in a neutral environment (pH = 7.4). The acid response of the POD-like activity of Zn_1–xMg_xFe₂O₄ ensured that the nanoenzyme would kill tumor cells only in the acidic tumor microenvironment, but would not damage normal cells. To further explore the influence of the Zn/Mg ratio on the POD-like catalytic efficiency of Zn_1–xMg_xFe₂O₄, the catalytic efficiency at 120 min was compared. As shown in Fig. 4C, the POD-like catalytic efficiency presented an earlier increasing and later decreasing trend with an increase of the Mg ratio; Zn_0.4Mg_0.6Fe₂O₄ and Zn_0.2Mg_0.8Fe₂O₄ had the highest POD-like catalytic efficiency.

Fig. 4 POD-like activity of Zn_0.4Mg_0.6Fe₂O₄. (A) ROS generation after treatment with Zn_1–xMg_xFe₂O₄ in an acidic environment. (B) Continuous ROS generation after treatment with Zn_0.4Mg_0.6Fe₂O₄ in different buffers (PBS, pH = 5.8 or 7.4). (C) Comparison of CAT-like activity at 120 min in an acidic environment.

2.2.3 Zn_0.4Mg_0.6Fe₂O₄ exhibited excellent GSH-like activity in an acid environment. Given the high H₂O₂ levels, OSCC also contained a large amount of GSH compared to normal tissues, which would increase the occurrence of chemotherapy tolerance.^16–18 Furthermore, previous research indicated that Fe³⁺ could be reduced to Fe²⁺ by GSH and Fe²⁺ could further convert H₂O₂ to O₂ or ROS.¹⁹ Therefore, the GSH-like activity of Zn_1–xMg_xFe₂O₄ was evaluated by detecting GSH consumption, Fe²⁺ generation, and Fe³⁺ depletion. As shown in Fig. 5A, GSH has been significantly consumed by Zn_0.4Mg_0.6Fe₂O₄ (14% at 6 h and 24% at 12 h) in an acidic environment. Furthermore, the generation of Fe²⁺ was also positively correlated with the concentration of Zn_0.4Mg_0.6Fe₂O₄ in an acid environment, while very little Fe²⁺ was generated in a neutral environment as shown in Fig. 5B, C and S3.† Furthermore, Fe³⁺ has also obviously been depleted after culture with GSH in acidic buffer, as shown in Fig. 5D. In summary, Zn_0.4Mg_0.6Fe₂O₄ exhibited the best enzyme-like activity among the Zn_1–xMg_xFe₂O₄ nanozymes which would increase sensitivity to chemotherapy.

Fig. 5 Properties of the GSH-like activity of Zn_0.4Mg_0.6Fe₂O₄. (A) GSH consumption after treatment with Zn_0.4Mg_0.6Fe₂O₄ in an acidic environment. (B) Fe²⁺ generation after treating with different concentrations of Zn_0.4Mg_0.6Fe₂O₄ in an acidic environment. (C) Fe²⁺ generation after treating with different concentrations of Zn_0.4Mg_0.6Fe₂O₄ in acidic and neutral environments. (D) Depletion of Fe³⁺ after treatment with Zn_0.4Mg_0.6Fe₂O₄.

2.3 Zn_0.4Mg_0.6Fe₂O₄ could shape the tumor microenvironment that inhibits the progression of OSCC

To further validate the CAT-like activity of Zn_0.4Mg_0.6Fe₂O₄in vitro, the levels of H₂O₂ and HIF-1α were detected in OSCC tumor cells treated with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄. As shown in Fig. 6A, compared to the normoxic environment, the H₂O₂ content was markedly elevated in tumor cells cultured in a hypoxic environment, and was also depleted after treatment with Zn_0.4Mg_0.6Fe₂O₄. The expression of HIF-1α reflected the O₂ content in the cells. As shown in Fig. 6B, the expression of HIF-1α in tumor cells cultured in a hypoxic environment was significantly higher than that in a normoxic environment and also decreased after treatment with Zn_0.4Mg_0.6Fe₂O₄. The ROS content was detected to validate the POD activity of Zn_0.4Mg_0.6Fe₂O₄in vitro. As shown in Fig. 6C, the ROS content in tumor cells was increased both in a normaxic and a hypoxic environment after treatment with Zn_0.4Mg_0.6Fe₂O_4., which further demonstrated that Zn_0.4Mg_0.6Fe₂O₄ also exhibited excellent enzyme-like activity in cells. Subsequently, a cell wound scratch assay and a transwell cell migration assay were performed to evaluate the influence of Zn_0.4Mg_0.6Fe₂O₄ on OSCC tumor cells. As shown in Fig. 6D and E, the migration rate of tumor cells increased significantly in the hypoxic environment compared to the normoxic environment, while the migration rate also decreased after treating with Zn_0.4Mg_0.6Fe₂O₄. This means that Zn_0.4Mg_0.6Fe₂O₄ may inhibit the progression of OSCC through an appropriate hypoxia environment.

Fig. 6 The influence of Zn_0.4Mg_0.6Fe₂O₄ on OSCC. (A) The effect of Zn_0.4Mg_0.6Fe₂O₄ on the H₂O₂ content, (B) on the expression of HIF-1α, (C) on ROS generation, and (D-E) on the migration ability of OSCC cells. **: P < 0.01, ***: P < 0.001.

2.4 Zn_0.4Mg_0.6Fe₂O₄ could improve the chemotherapy effect in OSCC

In addition to surgery, chemotherapy is also an important treatment for OSCC. However, due to the tolerance to chemotherapy caused by various factors, such as hypoxia, chemotherapy outcomes are still less than satisfactory. A chemosensitizer could enhance the effect of chemotherapy. Therefore, Zn_0.4Mg_0.6Fe₂O₄ was combined with DOX (first-line chemotherapy for OSCC) to treat OSCC in this study. First, the CCK-8 assay was performed to evaluate the toxicity of Zn_0.4Mg_0.6Fe₂O₄ to OSCC cells. As shown in Fig. 7A and B, cell viability decreased as the concentration of Zn_0.4Mg_0.6Fe₂O₄ increased in an acidic environment (IC₅₀-Zn_0.4Mg_0.6Fe₂O₄: 1.398 mg mL⁻¹) while no toxicity was observed in a neutral environment. Furthermore, the live/dead cell-double staining assay also showed greater OSCC cell death after treatment with 0.1 mg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ in an acidic environment, as shown in Fig. 7C, which indicated that the lethal effect of Zn_0.4Mg_0.6Fe₂O₄ also had acid-response properties in vitro. Subsequently, we further evaluated whether Zn_0.4Mg_0.6Fe₂O₄ could enhance the chemotherapy effect of DOX on OSCC cells. As shown in Fig. 7D, compared to DOX alone (IC₅₀-DOX: 0.737 mg mL⁻¹), OSCC cell viability obviously decreased when combined with 0.1 mg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ (IC₅₀-DOX and Zn_0.4Mg_0.6Fe₂O₄: 0.394 mg mL⁻¹ and 0.1 mg mL⁻¹, combination index: 0.616). Thus, Zn_0.4Mg_0.6Fe₂O₄ could efficiently enhance the chemotherapy effect of DOX on OSCC cells. Finally, a mouse xenograft OSCC tumor model was used to further validate our findings in vivo. As shown in Fig. 7E and F, the tumor volume of mice injected with Zn_0.4Mg_0.6Fe₂O₄ and DOX was significantly lower than that of mice injected with DOX alone, while the body weight of mice injected with Zn_0.4Mg_0.6Fe₂O₄ and DOX was higher as shown in Fig. 7G. Furthermore, cardiotoxicity was also less marked in mice injected with Zn_0.4Mg_0.6Fe₂O₄ compared to mice injected with DOX alone (Fig. 7H). Thus, Zn_0.4Mg_0.6Fe₂O₄ can not only enhance DOX chemotherapy, but can also efficiently reduce the toxicity of DOX chemotherapy in vivo. Therefore, Zn_0.4Mg_0.6Fe₂O₄ could serve as a chemo-sensitizer in the management of OSCC.

Fig. 7 Zn_0.4Mg_0.6Fe₂O₄ could enhance the chemotherapy effect in OSCC. CCK-8 assay showing the toxicity of different concentrations of Zn_0.4Mg_0.6Fe₂O₄ in OSCC cells (A) in a neutral environment and (B) in an acidic environment. (C) Double-staining assay for live/dead cells: toxicity of 0.1 mg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ in OSCC cells in neutral and acidic environments. (D) The influence of 0.1 mg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ on the toxicity of different concentrations of DOX in OSCC cells. (E and F) Comparison of in vivo therapeutic efficacy between DOX and DOX + Zn_0.4Mg_0.6Fe₂O₄. (G) Comparison of body weights of DOX and DOX + Zn_0.4Mg_0.6Fe₂O₄ treatments. (H) Comparison of in vivo systemic toxicity of DOX and DOX + Zn_0.4Mg_0.6Fe₂O₄. Note: ***: P < 0.001.

3 Experimental section

3.1 Synthesis of Zn_1–xMg_xFe₂O₄ nanozymes

The Zn_1–xMg_xFe₂O₄ nanozymes were prepared according to previously reported protocols with minor modifications.^20,21 Specifically, 0.05 M Mg(NO₃)₂·6H₂O/Zn(NO₃)₂·6H₂O (the ratio between Mg(NO₃)₂·6H₂O and Zn(NO₃)₂·6H₂O was established according to the stoichiometry of Zn_1–xMg_xFe₂O₄), 0.1 M Fe(NO₃)₃ 9H₂O and 0.15 M trisodium citrate were dissolved in 190 mL of distilled water and 10 mL of 1 M NaOH solution was then slowly added to the solution. The reactants were then placed in a 500-mL capacity container in a Teflon-lined autoclave. The autoclave was maintained at 160 °C for 6 h to prepare MgFe₂O₄, 180 °C for 8 h to prepare Zn_0.2Mg_0.8Fe₂O₄ and Zn_0.4Mg_0.6Fe₂O₄, and 180 °C for 6 h to prepare Zn_0.6Mg_0.4Fe₂O₄, Zn_0.8Mg_0.2Fe₂O₄, and ZnFe₂O₄.

3.2 Characterization of Zn_1–xMg_xFe₂O₄ nanozymes

The structural characterization and phase identifications of the sample are done with an X-ray diffractometer (Bruker AKS D8 Advance). The hydrodynamic diameter and zeta potential were measured using a dynamic light scattering instrument (DLS) (ZetaSizer Nano-ZS90; Malvern Instrument) in zeta potential analysis mode. The size and morphology of the obtained Zn_1–xMg_xFe₂O₄ nanozymes were observed by transmission electron microscopy (TEM; JEOL).

3.3 H₂O₂ consumption

The H₂O₂ consumption was measured using the colorimetric method of titanium sulfate (Ti(SO₄)₂) according to a previous protocol with minor modifications.²² Specifically, solution A: 2 mg of Zn_1–xMg_xFe₂O₄ was incubated with 5 mL of H₂O₂ (1 mM) in different buffers (pH 7.4 or pH 5.8) and stirred at 37 °C. Solution B: 665 μL of Ti(SO₄)₂ and 4.165 mL of H₂SO₄ were mixed with 25 mL of water. Then 100 μL of solution A was added to 200 μL of solution B at 1.5 h and 3 h. Ten minutes after addition, Zn_1–xMg_xFe₂O₄ was separated by centrifugation. UV-vis spectra were recorded to measure the remaining H₂O₂ by measuring the absorbance at 405 nm.

3.4 O₂ generation

The generation of O₂ was measured according to a method reported in the previous literature²² with minor modifications. In summary, 4 mg of Zn_1–xMg_xFe₂O₄ was incubated with 20 mL of PBS (pH = 5.8) containing 0.5 mM H₂O₂ at 37 °C. The dissolved O₂ concentration was monitored with an oxygen meter (HI9146, HANNA instruments, Korea) in real time at 60, 120, 180, 240, 300, 360, and 420 min.

3.5 POD-like activity detection

The TMB assay was used to detect the POD-like activity of Zn_1–xMg_xFe₂O₄. In brief, 20 μg of Zn_1–xMg_xFe₂O₄ was incubated in different buffers (pH 5.8 or pH 7.4) containing 1 mM TMB and 50 mM H₂O₂. POD-like activity was measured by detecting the absorbance at 625 nm at 30, 60, and 120 min.

3.6 GSH-like properties

GSH consumption, Fe²⁺ generation and Fe³⁺ depletion were investigated to detect GSH-like activity of Zn_0.4Mg_0.6Fe₂O₄.^19,22 To investigate the consumption of GSH, 475 μL of Zn_0.4Mg_0.6Fe₂O₄ (10 μg mL⁻¹) was incubated with 25 μL of GSH (4 mM) at 37 °C for 6 and 12 h. Zn_0.4Mg_0.6Fe₂O₄ was separated by centrifugation and the supernatant was collected for GSH measurement using the GSH kit (Beyotime Biotechnology). Finally, 10 μL of the supernatant was added to 100 μL of the reaction mixture from the GSH kit for 25 min and the concentration of GSH was measured by UV-vis spectroscopy at 410 nm. To investigate Fe²⁺ generation, different concentrations of Zn_0.4Mg_0.6Fe₂O₄ (0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 mg mL⁻¹) were incubated with/without 4 mM GSH in different buffers (pH 5.8 or pH 7.4) at 25 °C for 1 h, then the solutions were centrifuged (15 000 rpm, 20 min) and the supernatant was collected. A 100-μL volume of saturated 1, 10-phenanthroline solution was added to the supernatant. The absorbance at 512 nm was subsequently monitored. To investigate Fe³⁺ depletion, 10 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ was cultured with 4 mM GSH in acidic buffer (pH = 5.8) at 25 °C for 12, 24, 36, and 48 h. Then the Fe²⁺ generation was quantitatively analyzed according to the standard Fe²⁺ concentration absorbance curve, and the Fe³⁺ depletion was equal to the Fe³⁺ generation.

3.7 Cell culture

The OSCC cell line SCC9 was obtained from Fudan University (Shanghai, China); SCC9 cells were cultured in DMEM medium (Gibco) supplemented with 1% FBS (Sigma) in a humidified incubator at 37 °C with 5% CO₂. All the cell lines tested negative for mycoplasma contamination.

3.8 The detection of H₂O₂ concentration in tissues and cells

The H₂O₂ concentration in tissues and cells was measured using an H₂O₂ assay kit (S0038, Beyotime) according to the manufacturer's protocol. Specifically, for the detection of H₂O₂ in tumor tissues and normal tissues acquired from OSCC patients, tissue samples were first homogenized with lysis buffer supplied by the H₂O₂ assay kit (the ratio was 100 μL of lysate to 5 mg of tissues), and then centrifuged at 12 000 rpm for 5 min. All operations were performed on ice. The supernatant was collected for subsequent measurement of H₂O₂.

This study was conducted in accordance with the Declaration of Helsinki and was approved by the medical ethics committee of the Institute Affiliated Stomatology Hospital, Medical School of Nanjing University. Written informed consent was obtained from all patients. For extracellular and intracellular H₂O₂ measurement in tumor cells, SCC9 cells were first incubated in fresh culture medium in a normoxic environment (21% O₂), in fresh culture medium in a hypoxic environment (1% O₂), and in fresh culture medium supplemented with 100 μL of Zn_0.4Mg_0.6Fe₂O₄ in a hypoxic environment for 24 h. The culture medium was then harvested to determine the extracellular H₂O₂ concentration. Subsequently, cells were lysed in 100 μL of lysis buffer and supernatants, collected by centrifuging at 12 000×g for 10 min, and were used to determine the intracellular H₂O₂ concentration. The H₂O₂ concentration detection step was as follows: 50 μL of sample solution was incubated with 100 μL of reaction solution at room temperature for 30 min and then absorption at 560 nm was measured.

3.9 Detection of HIF-1α expression

HIF-1α protein expression was detected by the immunofluorescence assay. Specifically, 1 × 10³ cells (SCC9) were seeded in glass bottom culture plates. After 24 h of culture, cells were cultured in a normoxic environment, hypoxic environment, and hypoxic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ for an additional 24 h. The cells were then fixed with 4% paraformaldehyde for 20 min and blocked with 5% BSA for 30 min at room temperature. Cells were incubated with the primary antibody HIF-1α (Cat no.: 66730-1-Ig, Proteintech) overnight at 4 °C. Dylight-conjugated anti-mouse IgG (647-conjugated anti-mouse IgG, Abcam, USA) was used as the secondary antibody. The nucleus was stained with DAPI (Beyotime, China) and observed under a confocal scanning system (Ti, NIKON).

3.10 Intracellular ROS detection

Intracellular ROS expression was detected using a reactive oxygen species assay kit (Beyotime, China). In summary, 1 × 10³ cells (SCC9) were seeded in glass bottom culture plates. After 24 h of culture, cells were placed in a normoxic environment, a normoxic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄, a hypoxic environment, and in a hypoxic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ for an additional 24 h. The DCFH-DA probe (10 μM) was then added to the cells and incubated for another 30 min at 37 °C. Cells were washed three times with PBS and monitored using a fluorescence microscope (Ti2, NIKON) with 488 nm excitation.

3.11 Cell wound healing assay and transwell cell migration assay

The migration ability of OSCC cells was determined by the cell wound healing assay and the transwell cell migration assay. For the cell wound healing assay, Ibidi Culture-Insert 2 wells were placed in 6-well plates, and then SCC9 cells were seeded for 24 hours before forming a 500-μm cell scratch on the cell layer surface. Next, cells were cultured with a fresh medium in a normoxic environment, a hypoxic environment, and a hypoxic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄. Phase-contrast images were acquired at the time of the scratch and 24 h later. For the transwell cell migration assay, 100 μL of SCC9 was seeded in the upper chamber with 10% FBS in the lower chamber in a normoxic environment, a hypoxic environment, and a hypoxic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄. Twenty-four hours later, the cells at the bottom of the filter were stained with crystal violet staining solution (Beyotime, China), and the cell numbers were counted in five fields by microscopy.

3.12 Cell proliferation assay

The proliferation ability of OSCC cells was determined using a CCK-8 cell proliferation assay kit (Beyotime). Briefly, 1 × 10³ SCC9 cells were placed in a 96-well plate and cultured for 24 h. The culture medium was then replaced with fresh medium (pH 6.5 or pH 7.4) supplemented with a different concentration of Zn_0.4Mg_0.6Fe₂O₄ (0–1 mg mL⁻¹) or different concentrations of DOX (0–1 mg mL⁻¹). After 1 day of incubation, the culture medium was aspirated and treated cells were washed with PBS, before the addition of 10 μL of CCK8 solution to each well and incubated for an additional 2 hours at 37 °C. The absorbance of each well at 450 nm was then measured using an IMark enzyme mark instrument (Bio-Rad Inc., USA). The cell proliferation ability was calculated on the basis of the absorbance data.

3.13 Live/dead cell double staining assay

A Calcein-AM/PI double staining kit (Cat no.: 40747ES76, Yeasen, China) was used to detect live/dead cells. Briefly, 1 × 10³ cells (SCC9) were seeded in glass bottom culture plates. After 24 h of culture, cells were cultured in a neutral environment, in a neutral environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄, in an acidic environment, and in an acidic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ for an additional 24 h. The cells were then washed with 1× assay buffer three times and incubated with 100 μL of detection buffer (5 μL of 2 mM Calcein-AM solution and 15 μL of 1.5 mM PI solution in 5 mL of assay buffer) for 15 min at 3 °C and observed under a confocal scanning system.

3.14 Animal model

BALB/c athymic nude mice were provided by the Comparative Medical Center of YangZhou University. All mice were housed under SPF conditions in which food, water, bedding, and cages were irradiated prior to use. All animal procedures were performed according to Nanjing University Laboratory Animal Care and Use Guidelines and the experiments were approved by the Nanjing University Animal Ethics Committee. To investigate antitumor efficacy, mice with subcutaneous OSCC xenografts were randomly divided into three groups (n = 5 for each group) when the tumor reached approximately 0.05 cm³: (1) PBS group (intravenous injection of 100 μL of PBS), (2) DOX group (intravenous injection of 100 μL of PBS containing 0.25 mg kg⁻¹ DOX) and (3) DOX + Zn_0.4Mg_0.6Fe₂O₄ group (intravenous injection of 100 μL of PBS containing 0.25 mg kg⁻¹ DOX and 0.5 mg kg⁻¹ Zn_0.4Mg_0.6Fe₂O₄). The drugs were administered every 3 days for 5 times and the tumor volumes/body weight were recorded simultaneously. Tumor volumes were calculated using the following formula: V = 0.5ab² (a: the longest diameter and b: the shortest diameter). To examine histological changes in the organs of the experimental mice, including the heart, liver, spleen, lung and kidney, 15 days after drug administration, organs were collected from mice and samples were embedded in paraffin. Subsequently, the paraffin-embedded tissue sections were stained with H&E and observed using a light microscope.

3.15 Statistical analysis

Statistical analysis was performed using SPSS statistical software (version 23.0; IBM) and GraphPad Prism (Version 6.0, GraphPad Software, La Jolla, CA, USA). Student's two-sided t-test was used to evaluate statistical significance differences between two groups and one-way ANOVA was used to evaluate statistical significance differences between three or more groups. MANOVA was used to analyze the repeated measured data. A P-value ≤0.05 was considered statistically significant.

4 Conclusions

Zn_0.4Mg_0.6Fe₂O₄ possessed optimal enzyme-like activity including CAT-like, POD-like, and GSH-like activity across the series of Zn_1–xMg_xFe₂O₄ nanoenzymes in an acidic environment. Furthermore, Zn_0.4Mg_0.6Fe₂O₄ could effectively improve the efficacy of DOX chemotherapy in the treatment of OSCC and reduce cardiotoxicity. Zn_0.4Mg_0.6Fe₂O₄ could serve as a promising alternative chemosensitizer in the management of OSCC.

Author contributions

Liang Chen: conceptualization, data curation, and writing - original draft. Qingmei Kong: validation and formal analysis. Mingxing Tian: formal analysis and methodology. Qian Zhang: validation. Chengwan Xia: formal analysis and review & editing. Chao Deng: methodology, project administration, review & editing, funding acquisition, and supervision.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge support for this research from the Key Research Project Fund of Wannan Medical College (WK2019ZF07), Open Project of Key Laboratory of Non-coding RNA Transformation Research of Anhui higher education institutes (RNA202001), and Young Talents Training Project of Wannan Medical College (wyqnyx202008).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2na00750a

‡ These authors contributed equally to this article.

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