Biomimetic Nanozymes Suppressed Ferroptosis to Ameliorate Doxorubicin-Induced Cardiotoxicity via Synergetic Effect of Antioxidant Stress and GPX4 Restoration

Mitochondria-dependent ferroptosis plays an important role in the pathogenesis of doxorubicin (DOX)-induced cardiotoxicity (DIC), which remains a clinical challenge due to the lack of effective interventions. Cerium oxide (CeO2), a representative nanozyme, has attracted much attention because of its antioxidant properties. This study evaluated CeO2-based nanozymes for the prevention and treatment of DIC in vitro and in vivo by adding nanoparticles (NPs), which were synthesized by biomineralization, to the culture or giving them to the mice, and the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) was used as control. The prepared NPs exhibited an excellent antioxidant response and glutathione peroxidase 4 (GPX4)-depended bioregulation, with the additional merits of bio-clearance and long retention in the heart. The experiments showed that NP treatment could significantly reverse myocardial structural and electrical remodeling, and reduce myocardial necrosis. These cardioprotective therapeutic effects were associated with their ability to alleviate oxidative stress, mitochondrial lipid peroxidation, and mitochondrial membrane potential damage, with a superior efficiency to the Fer-1. The study also found that the NPs significantly restored the expression of GPX4 and mitochondrial-associated proteins, thereby restoring mitochondria-dependent ferroptosis. Therefore, the study provides some insights into the role of ferroptosis in DIC. It also shows that CeO2-based nanozymes could be a promising prevention and treatment candidate as a novel cardiomyocyte ferroptosis protector to mitigate DIC and improve prognosis and quality of life in cancer patients.


Synthesis of CeO2@BSA NPs
Based on previous studies [27,51], this could be biomineralized within the BSA protein as the template to assist the fabrication of Ce-based NPs. Ce(NO3)3·6H2O (9 mg) and BSA (60 mg) were dissolved in pure water (2.4 mL) at 37 °C and stirred for 5 min. NaOH (80 mM) was slowly added to the mixture drop by drop until the reaction system reached pH ≥ 10 to induce the formation of CeO2 and simultaneously unfold the BSA, which resulted in the precipitation of CeO2 nanoparticles into the BSA. The reaction solution was

Synthesis of CeO 2 @BSA NPs
Based on previous studies [27,51], this could be biomineralized within the BSA protein as the template to assist the fabrication of Ce-based NPs. Ce(NO 3 ) 3 ·6H 2 O (9 mg) and BSA (60 mg) were dissolved in pure water (2.4 mL) at 37 • C and stirred for 5 min. NaOH (80 mM) was slowly added to the mixture drop by drop until the reaction system reached pH ≥ 10 to induce the formation of CeO 2 and simultaneously unfold the BSA, which resulted in the precipitation of CeO 2 nanoparticles into the BSA. The reaction solution was then stirred at 55 • C for 8 h. The resulting CeO 2 @BSA were washed and separated by centrifugation at 12,000 rpm for 10 min to obtain a transparent supernatant. Ultrafiltration was also performed with a 30 kDa centrifugal filter unit [Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (30 kDa), Millipore] at 5000 rpm for 10 min to remove excess Ce 3+ in the supernatant. These purification procedures were repeated

Nanoparticles Characterization
The mean hydrodynamic size distribution was characterized using a BT-90 Nanoparticle Analyzer (Better, China) at 25 • C and the zeta potentials were monitored using NanoS90 (Malvern, Worcestershire, UK) at 25 • C. A drop of CeO 2 @BSA solution was carefully applied to the carbon-coated copper grids and dried under vacuum at room temperature. The morphology of the NPs was observed with high-resolution transmission electron microscopy (HRTEM, Philips Tecnai G2 F20, Amsterdam, The Netherlands). The crystal phase of CeO 2 @BSA was further confirmed via X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) was used to characterize the Ce hybrid oxidation state.

SOD Activity Assay and Catalase Activity Assay
The SOD-mimetic enzyme activity assay was measured according to the instructions of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The catalase (CAT)-mimetic enzyme activity assay was measured according to the methods published in the previous literature [28].

Cellular Uptake Experiments
H9c2 cells line (Cellcook Biotech Co., Ltd., Guangzhou, China) were pre-seeded into confocal Petri dishes and incubated in DMEM cell medium supplemented with 10% FBS at 37 • C in 5% CO 2 . When the cells entered the log phase growth, the medium was replaced with 1 mL of medium containing different concentrations of the CeO 2 @BSA-FITC NPs (200 µM). After a 6 h incubation period, the cells were washed three times with PBS and observed for a fluorescent signal using an Olympus FV1000 LSCM (Tokyo, Japan) under 488 nm for excitation and 525 nm for emission.

Cytotoxicity Assay
Standard MTT assays were carried out to visualize the cytotoxicity of CeO 2 @BSA. H9c2 cell lines were pre-seeded into 96-well cell culture plates. When the cells entered the log phase growth, the medium was replaced with 200 µL of medium containing different concentrations of the NPs (0, 50, 100, 150, 200, 300, 400, 500, 600, 800, 1000, or 2000 µM). After 24 h of incubation, the cells were washed three times with PBS, and MTT stock solution (5 mg/mL) was added to each well and left to react for 4 h. The medium was then discarded and dimethyl sulfoxide (150 µL/well, Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The assay plate was placed on a shaker (QLBE, Haimen, China) for 10 min, the optical density (OD) value for each well was recorded at 570 nm with a microplate reader (Thermo, Varioskan Flash). Cell viability was calculated using the following formula: cell viability (%) = (mean OD of selected treatment group/mean OD of the control group) × 100.
The data are presented as the mean value with standard deviation from six independent experiments.

Experimental Design and Model Establishment
C57BL/6J male mice aged 8-10 weeks (average body weight ≈ 25 g) were purchased from Beijing HuaFuKang Bioscience Co., Ltd. (Beijing, China). These mice were fed under controlled conditions with a constant temperature of 25 ± 2 • C, a humidity of 60 ± 5%, and a 12 h day/night cycle, given a sufficient standard animal diet and drinking water, and the cage bedding was changed every other day. All the animals were acclimated to the environment for one week before the experiment. The mice were randomly divided into four groups: control (NS, daily intraperitoneal administered normal saline, n = 6), DOX (twice every other day i.p. administered DOX, 7.5 mg/kg, n = 6), DOX + Fer-1 (daily i.p. administered Fer-1 1 mg/kg, n = 6), and DOX + CeO 2 @BSA NPs (intravenous administration 1 mg/kg measured by cerium for total of four doses, n = 6). The animal study protocol was approved by the Experimental Animal Management Committee of the Tianjin Medical University (TMUaMEC 2019004), and the experimental operation was carried out in accordance with the guidelines of the National Institutes of Health of the United States and in line with the 3R principles.

Determination of MDA, GSH, NT-proBNP, and cTnI in Mice Serum
The levels of malondialdehyde (MDA) and glutathione (GSH) were measured according to the instructions of the manufacturer (Solarbio, Beijing, China). Mice ELISA kits (Wuhan Huamei Biological Engineering Co., Ltd., Wuhan, China) were used to evaluate the serum levels of the N-terminal brain natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI).

Echocardiography
All the mice underwent transthoracic echocardiography utilizing a Vevo 2100 system with an MS400 linear array transducer (Visual Sonics, Toronto, ON, Canada) in a blinded manner by a specialist physician. Briefly, the mice were anesthetized with isoflurane, the chest hair was removed, and the mice were placed in a supine position on a constant temperature table at 37 • C, and a small animal ultrasound system was used to record transthoracic echocardiographic images. Two-dimensional and M-mode images of the longaxis and short-axis sections of the parasternal left ventricle of the mouse heart were collected, and the LVID, LVPW, and IVS were measured. The left ventricular ejection fractions (LVEF), left ventricular fractional shortening (LVFS), and left ventricular volume (LV Vol) were calculated according to international standards. All the ultrasound parameters were based on the average of 5 consecutive cardiac cycles.

Surface ECG and Epicardial Electrical Mapping
After satisfactory anesthesia with 1.5% tribromoethanol (20 mg/kg), at least 10 stable continuous electrocardiography (ECG) waveforms were recorded using a multiconductive physiological recorder (TOP-2001). Then, the widths of P wave, PR interval, QRS duration, QT interval, and RR interval were measured and analyzed using LabChart 8 Reader software (LabChart Pro software, version 8; AD Instruments, Shanghai, China). After surface ECG examination, with the support of a small animal ventilator, the chest was opened along the midline of the mouse chest, and on the epicardial surface of the left ventricle, a 6 × 6 electrode microelectrode (electrode impedance: 1.5-1.7Q, PA03606060101, multielectrode probe array) was used to record epicardial activation electrical mapping. Data were recorded using multichannel systems (EMS64-USB-1003, MappingLab Ltd., Oxford, UK). Date of the conduction velocity (CV), the inhomogeneity index, and the absolute inhomogeneity were calculated using EMapScope 4.0 software (MappingLab Ltd., Oxford, UK).

Blood Pressure
The mice were placed in animal holders on a far-infra-red warming platform, and their body temperatures were maintained at 37 • C. The blood pressure of conscious mice was measured using volume pressure-recording sensor equipment (BP-98-AL, Softron, Tokyo, Japan). After a period of stabilization, systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) were meticulously monitored.

Transmission Electron Microscope
The mouse left ventricular tissue samples were fixed in 2.5% glutaraldehyde (pH = 7.4) for 2 h. After being washed three times with 0.1 M phosphate buffer (pH = 7.2) and fixed in 1% osmic acid at 4 • C for 2 h, the samples were then gradient dehydrated in a graded series of ethanol. Subsequently, the samples were embedded in Epon-Araldite resin for penetration and placed in a model for polymerization. After the semi-thin section was used for positioning, the ultrathin section was made and collected for microstructure analysis, followed by the counterstaining of 3% uranyl acetate and 2.7% lead citrate. They were then observed with a HT7800 (HITACHI, Tokyo, Japan) transmission electron microscope.

Histology
After the ventricular tissue was wax-sealed and embedded in an automatic dehydrator, the tissue information was marked, and the 5 µm thick slice was maintained for serial sectioning. Selected intact and fold-free tissue sections were collected and laid flat in a spreader. After each tissue section was fully unfolded, it was quickly picked up with a glass slide, put in a 65 • C oven for 1 h, and then in the section box for preservation. The tissues were later stained to observe the morphology.

Cell Culture and Cell Experiments
The H9c2 cell line was used for the in vitro studies and maintained in a DMEM high-glucose culture medium (supplemented with 100 U/mL/100 mg/mL pen/strep) containing 10% FBS at 37 • C, and in a 5% CO 2 cell culture incubator with medium changes every other day. Cells were divided into four groups: control (PBS), DOX (1 µM for 24 h), DOX + Fer-1 (1 µM), and DOX + CeO 2 @BSA NPs (200 µM measured by cerium).

Cellular Mitochondrial Membrane Potential and ROS Measurement of H9c2 Cell Line
The H9c2 cells were incubated with JC-1 staining buffer in the dark for 20 min at 37 • C and washed twice with PBS. The processes of measurement were used with an Olympus FV1000 LSCM under the excitation wavelengths of 488 nm and 543 nm. The decreased relative fluorescence proportion of aggregate JC-1 (red)/monomeric JC-1 (green) indicates a decrease in the mitochondrial membrane potential (MMP). ROS were detected using a DCFH-DA fluorescent probe. The cells were incubated with 10 µM DCFH-DA in PBS in the dark for 20 min at 37 • C. Fluorescence was determined at 488 nm for excitation and 525 nm for emission. Microscope images were saved as TIFF files and processed for densitometric quantification with ImageJ version 1.46 (NIH). The software settings were kept the same for every image analyzed.

Mitochondrial Ferrous Ions and Mitochondrial Lipid Peroxidation Toxicity Assay
The H9c2 cells were incubated with Mito-FerroGreen (M489 Dojindo Laboratories, Kumamoto Prefecture, Japan) or MitoPeDPP (M466, Dojindo Laboratories, Kumamoto Prefecture, Japan) at 37 • C for 30 min out of the light. After incubation, the cells were

Biodistribution and Biocompatibility Assessment
The healthy C57BL/6J male mice were intravenously administered with CeO 2 @BSA normal saline solution (intravenous administration of 1 mg/kg measured by cerium for a total of four doses, n = 3) and the mice were sacrificed at the end of experiment. The major organs were collected, and then stained with HE to observe the morphological changes, and the remaining tissues were weighed and dissolved in a concentrated nitric acid solution. The amount of Ce elements in the different samples was analyzed by ICP-MS. In addition, blood samples were collected for hematological and biochemical analysis before the animals were euthanized.

Statistical Analysis
The data are presented as mean ± standard error of mean (SEM). The normal distribution was checked with the Kolmogorov-Smirnov test. Data corresponding to normal distributions were analyzed statistically using one-way analysis of variance (ANOVA), followed by the LSD test. Non-parametric tests were used for non-normally distributed data. The differences between the two groups were analyzed using the t test. All statistical analyses were performed using SPSS (version 26.0, International Business Machines Corporation, Armonk, NY, USA) or GraphPad Prism (version 8.0, GraphPad, San Diego, CA, USA) software for statistical charts and group charts, and p of < 0.05 was considered statistically significant.

CeO 2 @BSA NP Characterization
The CeO 2 @BSA NP solution had a clear and transparent brownish-yellow appearance after BSA biomineralization, though CeO 2 was insoluble in water ( Figure 2A). Transmission electron microscopy (TEM) images revealed the encapsulated CeO 2 @BSA was ≈ 3 nm in diameter (Figure 2A,B). The CeO 2 @BSA NPs showed an average hydrodynamic diameter of ≈14 nm from dynamic light scattering (DLS, Figure 2C). The stability of the CeO 2 @BSA NPs was evaluated by monitoring the NPs' size change using DLS at different time points at 4 • C ( Figure 2D), in different solvents ( Figure S1A), and at different pH levels ( Figure S1B). Next, the zeta potential of the BSA remained stable (from −6.627 mV to −32.40 mV, Figure 2E) after the reaction. Moreover, for phase analysis, the XRD pattern analysis ( Figure 2F) exhibited three prominent peaks characteristic of cerium oxide, corresponding to the (111)-, (220)-, and (311)-planes (JCPDS: No.01-078-3080), respectively, in the cubic fluorite crystal structure. This suggests the existence of CeO 2 within the BSA protein templates. XPS revealed the hybrid nature of Ce (III) to Ce (IV) ions, with corresponding binding energy peaks for Ce 3+ (885.00 ev and 903.50 ev) and Ce 4+ (882.10 ev, 888.10 ev, 898.00 ev, 900.90 ev, 906.40 ev, and 916.35 ev) in the spectrum of the Ce3d (Figure 2G), and the ratio between them was roughly 1:3, which almost coincided with the spectrum of a previous study [51]. Meanwhile, the spectrum of the O1s ( Figure 2H) showed many oxygen vacancies in CeO 2 @BSA NPs, which enhanced their effectiveness in catalyzing ROS. nm in diameter (Figure 2A,B). The CeO2@BSA NPs showed an average hydrodynamic diameter of ≈14 nm from dynamic light scattering (DLS, Figure 2C). The stability of the CeO2@BSA NPs was evaluated by monitoring the NPs' size change using DLS at different time points at 4 °C ( Figure 2D), in different solvents ( Figure S1A), and at different pH levels ( Figure S1B). Next, the zeta potential of the BSA remained stable (from −6.627 mV to −32.40 mV, Figure 2E) after the reaction. Moreover, for phase analysis, the XRD pattern analysis ( Figure 2F) exhibited three prominent peaks characteristic of cerium oxide, corresponding to the (111)-, (220)-, and (311)-planes (JCPDS: No.01-078-3080), respectively, in the cubic fluorite crystal structure. This suggests the existence of CeO2 within the BSA protein templates. XPS revealed the hybrid nature of Ce (III) to Ce (IV) ions, with corresponding binding energy peaks for Ce 3+ (885.00 ev and 903.50 ev) and Ce 4+ (882.10 ev, 888.10 ev, 898.00 ev, 900.90 ev, 906.40 ev, and 916.35 ev) in the spectrum of the Ce3d (Figure 2G), and the ratio between them was roughly 1:3, which almost coincided with the spectrum of a previous study [51]. Meanwhile, the spectrum of the O1s ( Figure 2H) showed many oxygen vacancies in CeO2@BSA NPs, which enhanced their effectiveness in catalyzing ROS.

In Vitro Hybrid Enzymatic Mimetic Activity of CeO2@BSA NPs
We next found that CeO2@BSA NPs had excellent SOD-mimetic ( Figure 3A) and CAT-hybrid-mimetic enzyme (Figures 3B and S2) activities, which were dose-dependent in vitro. The Ce 3+ redox state is peculiar to colorless ions and the coloration is peculiar to the Ce 4+ redox state. A color change in the NP solution after the addition of hydrogen peroxide indicates a change in the Ce 4+ /Ce 3+ state, showing that a redox reaction took place

In Vitro Hybrid Enzymatic Mimetic Activity of CeO 2 @BSA NPs
We next found that CeO 2 @BSA NPs had excellent SOD-mimetic ( Figure 3A) and CAT-hybrid-mimetic enzyme (Figures 3B and S2) activities, which were dose-dependent in vitro. The Ce 3+ redox state is peculiar to colorless ions and the coloration is peculiar to the Ce 4+ redox state. A color change in the NP solution after the addition of hydrogen peroxide indicates a change in the Ce 4+ /Ce 3+ state, showing that a redox reaction took place [28]. After ten days, the solution turned colorless and another color change took place after the addition of hydrogen peroxide, demonstrating the existence of Ce 4+ /Ce 3+ redox cycling.
Nutrients 2023, 15, x FOR PEER REVIEW 9 of 20 [28]. After ten days, the solution turned colorless and another color change took place after the addition of hydrogen peroxide, demonstrating the existence of Ce 4+ /Ce 3+ redox cycling.

CeO2@BSA NPs Protected DIC by Preventing Ferroptosis in Cardiomyocytes
To further evaluate the potential for biomedical applications, the methyl thiazolyl tetrazolium (MTT) assay showed that the viability of H9c2 cells were not influenced by CeO2@BSA NPs, even at high doses of Ce of up to 200 μM ( Figure 4A). This suggested the great biocompatibility of the NPs. We confirmed that the CeO2@BSA NPs were taken up by the H9c2 cells in large quantities ( Figure 4B). Additionally, the MTT assay showed that 1 μM DOX significantly inhibited cellular activity after a 24 h co-incubation ( Figure S3).
The loss of cardiomyocytes is the major cause of DOX-induced cardiac injury and dysfunction. Therefore, the prevention of cardiomyocyte loss was an effective approach to the treatment of DIC [52]. DCFH-DA fluorescence found that the intracellular ROS level was significantly higher in the DOX group compared with the PBS group and the ROS level was markedly decreased both in the CeO2@BSA and Fer-1 group ( Figure 4C,G). Mitochondrial membrane potential (MMP, ΔΨm) is commonly used to measure mitochondrial function, and a loss of ΔΨm indicated mitochondrial dysfunction [53]. MMP was quantified using the JC-1 dye. The MMP was significantly reduced in the DOX group and improved both in the CeO2@BSA and Fer-1 group ( Figure 4D,H).
Ferroptosis was distinctly different from other known cell death modalities in terms of cell morphology, genetics, and biochemistry. The main feature of ferroptosis was an iron-dependent lethal accumulation of lipid peroxidation and ROS accumulation. Free intracellular iron ions were in the form of stable Fe 2+ and Fe 3+ . Mito-FerroGreen is a new fluorescent probe for the detection of the ferrous ion (Fe 2+ ) in mitochondria, which is the site of iron-sulfur clusters and hemoglobin synthesis. Mito-FerroGreen fluorescence was significantly strong in the DOX group and markedly decreased in the CeO2@BSA group, but Fer-1 did not significantly reverse the production of mitochondrial ferrous ions (Figure 4E,I). MitoPeDPP is a novel fluorescent dye that could cross cell membranes and accumulate on the inner mitochondrial membrane due to its triphenylphosphine structure, which could be oxidized by lipid peroxides to emit strong fluorescence, indicating the occurrence of mitochondria-dependent ferroptosis [31]. MitoPeDPP fluorescence was significantly strong in the DOX group and markedly decreased both in the CeO2@BSA and Fer-1 groups ( Figure 4F,J). The above results shows that the CeO2@BSA NPs could decrease ROS, suppress ferroptosis, and improve mitochondrial function at the cellular level. The results of the study also showed that the CeO2@BSA NPs outperformed Fer-1.

CeO 2 @BSA NPs Protected DIC by Preventing Ferroptosis in Cardiomyocytes
To further evaluate the potential for biomedical applications, the methyl thiazolyl tetrazolium (MTT) assay showed that the viability of H9c2 cells were not influenced by CeO 2 @BSA NPs, even at high doses of Ce of up to 200 µM ( Figure 4A). This suggested the great biocompatibility of the NPs. We confirmed that the CeO 2 @BSA NPs were taken up by the H9c2 cells in large quantities ( Figure 4B). Additionally, the MTT assay showed that 1 µM DOX significantly inhibited cellular activity after a 24 h co-incubation ( Figure S3).
The loss of cardiomyocytes is the major cause of DOX-induced cardiac injury and dysfunction. Therefore, the prevention of cardiomyocyte loss was an effective approach to the treatment of DIC [52]. DCFH-DA fluorescence found that the intracellular ROS level was significantly higher in the DOX group compared with the PBS group and the ROS level was markedly decreased both in the CeO 2 @BSA and Fer-1 group ( Figure 4C,G). Mitochondrial membrane potential (MMP, ∆Ψm) is commonly used to measure mitochondrial function, and a loss of ∆Ψm indicated mitochondrial dysfunction [53]. MMP was quantified using the JC-1 dye. The MMP was significantly reduced in the DOX group and improved both in the CeO 2 @BSA and Fer-1 group ( Figure 4D,H).
Ferroptosis was distinctly different from other known cell death modalities in terms of cell morphology, genetics, and biochemistry. The main feature of ferroptosis was an iron-dependent lethal accumulation of lipid peroxidation and ROS accumulation. Free intracellular iron ions were in the form of stable Fe 2+ and Fe 3+ . Mito-FerroGreen is a new fluorescent probe for the detection of the ferrous ion (Fe 2+ ) in mitochondria, which is the site of iron-sulfur clusters and hemoglobin synthesis. Mito-FerroGreen fluorescence was significantly strong in the DOX group and markedly decreased in the CeO 2 @BSA group, but Fer-1 did not significantly reverse the production of mitochondrial ferrous ions ( Figure 4E,I). MitoPeDPP is a novel fluorescent dye that could cross cell membranes and accumulate on the inner mitochondrial membrane due to its triphenylphosphine structure, which could be oxidized by lipid peroxides to emit strong fluorescence, indicating the occurrence of mitochondria-dependent ferroptosis [31]. MitoPeDPP fluorescence was significantly strong in the DOX group and markedly decreased both in the CeO 2 @BSA and Fer-1 groups ( Figure 4F,J). The above results shows that the CeO 2 @BSA NPs could decrease ROS, suppress ferroptosis, and improve mitochondrial function at the cellular level. The results of the study also showed that the CeO 2 @BSA NPs outperformed Fer-1.

Preventive and Therapeutic Efficacy of CeO 2 @BSA NPs on Doxorubicin-Induced Cardiac Structural and Electrical Remodeling in Mice
The cardiotoxicity of DOX was mainly related to its cumulative dose of the drug and we performed mice modelling using a cumulative dose of 15 mg/kg, as recommended by the European Society of Clinical Oncology (ESMO) guidelines. The preventive and therapeutic interventions of CeO 2 @BSA NPs were administered through the tail vein and Fer-1 was administered intraperitoneally. The schematic animal experiment protocol is shown in Figure 5A. There was a significant decrease in body weight and a poor general condition of the mice in the DOX group, which was improved by the administration of Fer-1 and CeO 2 @BSA NPs ( Figure 5B). Hemodynamics through the non-invasive monitoring of mice tails showed that the HR, SBP, DBP, and MBP were not significantly different between the four groups ( Figure S4).
Echocardiograms showed abnormality of diastolic and systolic functions ( Figure S5) in the DOX group, manifested by a decrease in the left ventricular ejection fraction (LVEF, Figure 5C), a decrease in the left ventricular fractional shortening (LVFS, Figure 5D), a larger left ventricular internal diameter (LVID, Figure 5E), and an expanded left ventricular volume (LV Vol, Figure 5F), which could be reversed by Fer-1 and CeO 2 @BSA NPs.
For the ECG data, there was a significantly longer PR interval in the DOX group and recovery in CeO 2 @BSA NPs ( Figure 5G), despite the P wave duration, QRS duration, and QT and RR interval being not significantly different across the four experimental groups ( Figure S6).
Serum cTnI and NT-proBNP are recognized markers of cardiomyocyte and cardiac impairment [32]. They were significantly elevated in the DOX group, and decreased in the treatment group ( Figure 5H,I).
In vivo epicardial electrical labelling (mapping images, Figure 5J) showed that the left ventricular conduction velocity (LVCV, Figure 5K) was significantly lower in the DOX group, accompanied by higher LV conduction dispersion (absolute, Figure 5L; index, Figure 5M), which was consistent with prior clinical trial findings [54] and may be the basis of malignant ventricular arrhythmias caused by DOX. However, the above abnormalities of cardiac electrical conduction were restored by Fer-1 and CeO 2 @BSA NPs, though CeO 2 @BSA NPs were significantly superior to Fer-1.
Glutathione is a tripeptide compound in the body that is involved in antioxidant and drug metabolism and is usually present in a reduced state (GSH) [33]. MDA is a compound formed after the breakdown of lipid peroxides [55]. Both are widely used as markers of oxidative stress and ferroptosis. In line with previous studies, GSH was significantly lower in the DOX group and recovered significantly in the CeO 2 @BSA NP group, but did not reach statistical significance in the Fer-1 group ( Figure 5N). Meanwhile, MDA was significantly higher in the DOX group and reduced significantly in both treated groups ( Figure 5O).
Hematoxylin/eosin (HE) staining was used to evaluate morphological changes in the myocardium. The results showed that compared with the NS group, the myocardial structure of the ventricular tissue in the DOX group was destroyed, and the cardiomyocytes were extensively vacuolated and arranged in a disorderly manner. All these abnormal changes were, however, reversed in the CeO 2 @BSA and Fer-1 groups ( Figure 5P).
Combining the above results, CeO 2 @BSA NPs could be a promising treatment candidate for reversing the cardiac structural and electrical remodeling caused by DIC. A recent study has reported the use of non-drug interventions for the treatment of DIC. Prathumsap et al. [56] reported that vagus nerve stimulation (VNS) exerted cardioprotective effects against DIC via the activation of both the muscarinic receptor (mAChR) and nicotinic acetylcholine receptor (nAChR). However, large clinical trials are needed to further verify the safety and efficacy. Data are expressed as mean ± SEM of three to six independent replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns: no statistical difference.

CeO2@BSA NPs Exhibited Protective Effect against Ferroptosis by Restoring GPX4 Expression and Improving Mitochondrial Function and Homeostasis
Mitochondria are important organelles in the DOX-induced ferroptosis of cardiomyocytes [26,57]. Oh et al. reported that mitochondrial events could be the ultimate step in determining the final cell fate [58]. Smaller mitochondria, increased bilayer density, and Data are expressed as mean ± SEM of three to six independent replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns: no statistical difference.

CeO 2 @BSA NPs Exhibited Protective Effect against Ferroptosis by Restoring GPX4 Expression and Improving Mitochondrial Function and Homeostasis
Mitochondria are important organelles in the DOX-induced ferroptosis of cardiomyocytes [26,57]. Oh et al. reported that mitochondrial events could be the ultimate step in determining the final cell fate [58]. Smaller mitochondria, increased bilayer density, and the reduced cristae of mitochondria in cardiomyocytes were observed by TEM in the DOX group, suggesting mitochondrial-dependent ferroptosis that was consistent with previous studies [25,59]. Additionally, the mitochondrial injury was, however, reversed by Fer-1 and CeO 2 @BSA NPs ( Figure 6A).
The GPX4 attenuated lipid peroxide toxicity and maintained membrane lipid bilayer homeostasis to inhibit ferroptosis due to its catalytic activity. The inactivation of GPX4, however, led to a disrupted oxidative balance and disrupted membrane structure, provoking ferroptosis. Because of its specific mechanism of action, GPX4 was considered a ferroptosis core regulator [60]. Consistent with this, we evaluated the expression of GPX4 and the mitochondria-associated protein of the myocardial tissues and found that the expression levels of GPX4, mtTFA, PGC-1α, and DRP1 were significantly lower in the DOX group compared with the NS group ( Figure 6B-I). The above abnormalities were, however, significantly restored in the CeO 2 @BSA and Fer-1 groups, except for GPX4 protein expression which was not statistically different compared to the Fer-1 group.
Mitochondrial homeostasis is important for maintaining the normal functions of mitochondria [61,62]. The mtTFA protein is bound to the mitochondrial light strand promoter to participate in mitochondrial transcription regulation, which is required for the maintenance of normal levels of mitochondrial DNA [63]. The PGC-1α is a master controller that promotes the transcription of the genes involved in mitochondrial biogenesis and oxidative metabolism, hence enhancing fatty acid β-oxidation, oxidative phosphorylation, and ATP production [64]. The DRP1 is required for mitochondrial fission during mitosis and plays a role in mitochondrial and peroxisomal division [65].
Recently, Wang et al. [66] reported that DOX can significantly affect the pathways related to the energy metabolism of myocardial cells via metabonomic analysis. Additionally, Di'ao Xinxuekang (DXXK) could improve DIC in mice by inhibiting ferroptosis through the AMPK-mediated energy protection pathway. He et al. [67] reported that epigallocate-chin-3-gallate pretreatment could effectively decrease iron accumulation, inhibit oxidative stress and abnormal lipid metabolism, and thereby alleviate DIC ferroptosis by upregulating AMPKα2 and activating adaptive autophagy. These results suggest that our future research should focus more on mitochondrial energy metabolism.
To summarize, the above results reveal that the cardioprotective effects of CeO 2 @BSA NPs against DIC may be mediated by the restoring of GPX4 expression, thereby maintaining mitochondrial homeostasis and function, as well as restoring mitochondria-dependent ferroptosis. The results of the study also provide some insights into the role of ferroptosis in DIC.

In Vivo Metabolism and Biocompatibility of CeO 2 @BSA NPs
The biocompatibility of CeO 2 @BSA NPs was also evaluated systematically following its administration via the tail vein. The cerium content in the major organs was determined via ICP-MS analysis. The metabolism of CeO 2 @BSA NPs in the heart showed a persistent presence compared to the equivalent cerium content of Ce(NO 3 ) 3 ( Figures 7A and S7), suggesting a high stability provided by biomimetic mineralization. Meanwhile, the metabolism of cerium in the major tissues and organs showed that it was mainly metabolized by the hepatobiliary systems ( Figure S8) in line with a prior study [68]. The routine blood examination ( Figure 7B-F), liver function test ( Figure 7G-I), renal function test ( Figure 7J-K), morphological changes in the major organs ( Figure 7L), and absence of obvious bodyweight loss ( Figure S9) after a week post-injection showed that CeO 2 @BSA has excellent biocompatibility and low toxicity. This could be exploited to develop a novel cardiomyocyte ferroptosis protector to mitigate DIC in cancer patients and improve their prognosis and quality of life. CeO2@BSA nanoparticles, GPX4: glutathione peroxidase 4, mtTFA: mitochondrial transcription factor A, PGC-1α: α subunit of peroxisome proliferators-activated receptor-γcoactivator-1, DRP1: dynamin-related protein 1. Data are expressed as mean ± SEM of three to five independent replicates; * p < 0.05, *** p < 0.001, **** p < 0.001, ns: no statistical difference.

In Vivo Metabolism and Biocompatibility of CeO2@BSA NPs
The biocompatibility of CeO2@BSA NPs was also evaluated systematically following its administration via the tail vein. The cerium content in the major organs was determined via ICP-MS analysis. The metabolism of CeO2@BSA NPs in the heart showed a persistent presence compared to the equivalent cerium content of Ce(NO3)3 ( Figures 7A  and S7), suggesting a high stability provided by biomimetic mineralization. Meanwhile, the metabolism of cerium in the major tissues and organs showed that it was mainly metabolized by the hepatobiliary systems ( Figure S8) in line with a prior study [68]. The routine blood examination ( Figure 7B-F), liver function test ( Figure 7G-I), renal function test ( Figure 7J-K), morphological changes in the major organs ( Figure 7L), and absence of obvious body-weight loss ( Figure S9) after a week post-injection showed that CeO2@BSA has excellent biocompatibility and low toxicity. This could be exploited to develop a novel cardiomyocyte ferroptosis protector to mitigate DIC in cancer patients and improve their prognosis and quality of life. . Representative HE staining of heart, liver, spleen, lung, kidney, and brain in the two groups after a week of treatment, scale bar = 100 μm. WBC: white blood cell, RBC: red blood cell, ALT: alanine transaminase, AST: aspartate transaminase, ALP: alkaline phosphatase, . Representative HE staining of heart, liver, spleen, lung, kidney, and brain in the two groups after a week of treatment, scale bar = 100 µm. WBC: white blood cell, RBC: red blood cell, ALT: alanine transaminase, AST: aspartate transaminase, ALP: alkaline phosphatase, Cre: creatinine, BUN: blood urea nitrogen, HE staining: hematoxylin/eosin (HE). Data are expressed as mean ± SEM of three independent replicates; ** p < 0.01, **** p < 0.001, ns: no statistical difference.

Study Limitations
There are some limitations associated with the present study. Firstly, we did not block the GPX4 pathway of mitochondrial oxidation in vivo or in vitro; thus, we could not further clarify that it was the sole signaling pathway to improve mitochondrial function by CeO 2 @BSA NPs. Secondly, we only assessed the effects of CeO 2 @BSA NPs on cardiomyocytes, whereas the potential effects of NPs on fibroblasts, immunocytes, mesothelium, endotheliocytes, or intercellular communication were not investigated [69][70][71][72].

Conclusions
The current study showed that the biomimetic mineralization of hybrid CeO 2 -based nanozymes against DIC were mediated by the effect of antioxidant stress and the restoration of GPX4 expression, thereby maintaining mitochondrial homeostasis and function, and restoring mitochondria-dependent ferroptosis. This provides some insights into the role of ferroptosis in DIC. Furthermore, the nanozyme could be a promising prevention and treatment candidate for clinical translation as a novel cardiomyocyte ferroptosis protector to mitigate DIC and improve the prognosis and quality of life of cancer patients.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nu15051090/s1, Figure S1: Particle size of CeO 2 @BSA nanoparticles in different solvents and different pH levels measured by dynamic light scattering; Figure S2: Digital images of catalase mimetic enzyme of CeO 2 @BSA nanoparticles; Figure S3: The effect of DOX on cellular viability; Figure S4: Analysis of the results of hemodynamics through noninvasive monitoring of mice tail; Figure S5: Representative electrocardiography recorded for the mice; Figure S6: Representative electrocardiography recorded for the mice in the four groups and analysis results of the P wave duration, QRS duration, QT interval, and RR interval. Figure S7: Metabolism of CeO 2 @BSA nanoparticles in the heart at different times of the day; Figure S8: Metabolism of CeO 2 @BSA nanoparticles in the major tissues and organs at different times of the week; Figure S9: Body-weight change in CeO 2 @BSA nanoparticles and normal saline groups.