Zn0.4Mg0.6Fe2O4 nanoenzyme: a novel chemo-sensitizer for the chemotherapy treatment of oral squamous cell carcinoma

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 Zn1–xMgxFe2O4 nanomaterials with enzyme-like properties, including catalase (CAT)-like, peroxidase (POD)-like, and glutathione (GSH)-like activity in an acidic environment. Among them, Zn0.4Mg0.6Fe2O4 performed the best and effectively increased the efficacy of doxorubicin (DOX) chemotherapy for the treatment of OSCC with reduced cardiotoxicity. Therefore, Zn0.4Mg0.6Fe2O4 could serve as a novel chemosensitizer in the treatment of OSCC.


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 inltration 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 eld. 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 MnFe 2 O 4 could catalyze H 2 O 2 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][14][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-x Mg x Fe 2 O 4 nanozymes were synthesized and, of these, Zn 0.4 Mg 0.6 Fe 2 O 4 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 0.4 Mg 0.6 Fe 2 O 4 were veried in vitro. Finally, we veried whether Zn 0.4 Mg 0.6 Fe 2 O 4 could reduce chemotherapy tolerance and improve the effect of chemotherapy on OSCC, and the results showed that Zn 0.4 Mg 0.6 Fe 2 O 4 could serve as a promising chemosensitizer in OSCC management. The main scheme of our study is depicted below (Fig. 1 A series of Zn 1-x Mg x Fe 2 O 4 molecules were successfully synthesized as shown in Fig. 2A and the X-ray diffraction (XRD) spectra are given in Fig. 2B 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   Given the high H 2 O 2 levels, OSCC also contained a large amount of GSH compared to normal tissues, which would increase the occurrence of chemotherapy tolerance. [16][17][18] Furthermore, previous research indicated that Fe 3+ could be reduced to Fe 2+ by GSH and Fe 2+ could further convert H 2 O 2 to O 2 or ROS. 19 Therefore, the GSH-like activity of Zn 1-x Mg x Fe 2 O 4 was evaluated by detecting GSH consumption, Fe 2+ generation, and Fe 3+ depletion. As shown in Fig. 5A, GSH has been signicantly consumed by Zn 0.4 Mg 0.6 Fe 2 O 4 (14% at 6 h and 24% at 12 h) in an acidic environment. Furthermore, the generation of Fe 2+ was also positively correlated with the concentration of Zn 0.4 Mg 0.6 Fe 2 O 4 in an acid environment, while very little Fe 2+ was generated in a neutral environment as shown in Fig. 5B, C and S3. † Furthermore, Fe 3+ has also obviously been depleted aer culture with GSH in acidic buffer, as shown in  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 0.4 Mg 0.6 Fe 2 O 4 was combined with DOX (rst-line chemotherapy for OSCC) to treat OSCC in this study. First, the CCK-8 assay was performed to evaluate the toxicity of Zn 0.4 -Mg 0.6 Fe 2 O 4 to OSCC cells. As shown in Fig. 7A  cardiotoxicity was also less marked in mice injected with Zn 0.4 Mg 0.6 Fe 2 O 4 compared to mice injected with DOX alone (Fig. 7H)

Characterization of Zn 1-x Mg x Fe 2 O 4 nanozymes
The structural characterization and phase identications 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-x Mg x Fe 2 O 4 nanozymes were observed by transmission electron microscopy (TEM; JEOL).

H 2 O 2 consumption
The H 2 O 2 consumption was measured using the colorimetric method of titanium sulfate (Ti(SO 4 ) 2 ) according to a previous protocol with minor modications. 22

O 2 generation
The generation of O 2 was measured according to a method reported in the previous literature 22 with minor modications. In summary, 4 mg of Zn 1-x Mg x Fe 2 O 4 was incubated with 20 mL of PBS (pH = 5.8) containing 0.5 mM H 2 O 2 at 37°C. The dissolved O 2 concentration was monitored with an oxygen meter (HI9146, HANNA instruments, Korea) in real time at 60, 120, 180, 240, 300, 360, and 420 min.

POD-like activity detection
The TMB assay was used to detect the POD-like activity of Zn 1- x Mg x Fe 2 O 4 . In brief, 20 mg of Zn 1-x Mg x Fe 2 O 4 was incubated in different buffers (pH 5.8 or pH 7.4) containing 1 mM TMB and 50 mM H 2 O 2 . POD-like activity was measured by detecting the absorbance at 625 nm at 30, 60, and 120 min.

GSH-like properties
GSH consumption, Fe 2+ generation and Fe 3+ depletion were investigated to detect GSH-like activity of Zn 0.4 Mg 0.6 Fe 2 O 4 . 19,22 To investigate the consumption of GSH, 475 mL of Zn 0.4 Mg 0.6 -Fe 2 O 4 (10 mg mL −1 ) was incubated with 25 mL of GSH (4 mM) at 37°C for 6 and 12 h. Zn 0.4 Mg 0.6 Fe 2 O 4 was separated by centrifugation and the supernatant was collected for GSH measurement using the GSH kit (Beyotime Biotechnology). Finally, 10 mL of the supernatant was added to 100 mL 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 Fe 2+ generation, different concentrations of Zn 0.4 Mg 0.6 Fe 2 O 4 (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-mL volume of saturated 1, 10-phenanthroline solution was added to the supernatant. The absorbance at 512 nm was subsequently monitored. To investigate Fe 3+ depletion, 10 mg mL −1 Zn 0.4 -Mg 0.6 Fe 2 O 4 was cultured with 4 mM GSH in acidic buffer (pH = 5.8) at 25°C for 12, 24, 36, and 48 h. Then the Fe 2+ generation was quantitatively analyzed according to the standard Fe 2+ concentration absorbance curve, and the Fe 3+ depletion was equal to the Fe 3+ generation.

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 humidied incubator at 37°C with 5% CO 2 . All the cell lines tested negative for mycoplasma contamination.

The detection of H 2 O 2 concentration in tissues and cells
The H 2 O 2 concentration in tissues and cells was measured using an H 2 O 2 assay kit (S0038, Beyotime) according to the manufacturer's protocol. Specically, for the detection of H 2 O 2 in tumor tissues and normal tissues acquired from OSCC patients, tissue samples were rst homogenized with lysis buffer supplied by the H 2 O 2 assay kit (the ratio was 100 mL 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 H 2 O 2 .
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 H 2 O 2 measurement in tumor cells, SCC9 cells were rst incubated in fresh culture medium in a normoxic environment (21% O 2 ), in fresh culture medium in a hypoxic environment (1% O 2 ), and in fresh culture medium supplemented with 100 mL of Zn 0.4 Mg 0.6 Fe 2 O 4 in a hypoxic environment for 24 h. The culture medium was then harvested to determine the extracellular H 2 O 2 concentration. Subsequently, cells were lysed in 100 mL of lysis buffer and supernatants, collected by centrifuging at 12 000×g for 10 min, and were used to determine the intracellular H 2 O 2 concentration. The H 2 O 2 concentration detection step was as follows: 50 mL of sample solution was incubated with 100 mL of reaction solution at room temperature for 30 min and then absorption at 560 nm was measured.

Detection of HIF-1a expression
HIF-1a protein expression was detected by the immunouorescence assay. Specically, 1 × 10 3 cells (SCC9) were seeded in glass bottom culture plates. Aer 24 h of culture, cells were cultured in a normoxic environment, hypoxic environment, and hypoxic environment supplemented with 100 mg mL −1 Zn 0.4 Mg 0.6 Fe 2 O 4 for an additional 24 h. The cells were then xed 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-1a (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).

Intracellular ROS detection
Intracellular ROS expression was detected using a reactive oxygen species assay kit (Beyotime, China). In summary, 1 × 10 3 cells (SCC9) were seeded in glass bottom culture plates. Aer 24 h of culture, cells were placed in a normoxic environment, a normoxic environment supplemented with 100 mg mL −1 Zn 0.4 Mg 0.6 Fe 2 O 4 , a hypoxic environment, and in a hypoxic environment supplemented with 100 mg mL −1 Zn 0.4 Mg 0.6 Fe 2 O 4 for an additional 24 h. The DCFH-DA probe (10 mM) 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 uorescence microscope (Ti2, NIKON) with 488 nm excitation.

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-mm 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 mg mL −1 Zn 0.4 Mg 0.6 Fe 2 O 4 . Phase-contrast images were acquired at the time of the scratch and 24 h later. For the transwell cell migration assay, 100 mL 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 mg mL −1 Zn 0.4 Mg 0.6 Fe 2 O 4 . Twenty-four hours later, the cells at the bottom of the lter were stained with crystal violet staining solution (Beyotime, China), and the cell numbers were counted in ve elds by microscopy.

Cell proliferation assay
The proliferation ability of OSCC cells was determined using a CCK-8 cell proliferation assay kit (Beyotime). Briey, 1 × 10 3 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 0.4 Mg 0.6 Fe 2 O 4 (0-1 mg mL −1 ) or different concentrations of DOX (0-1 mg mL −1 ). Aer 1 day of incubation, the culture medium was aspirated and treated cells were washed with PBS, before the addition of 10 mL 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.