Cerium (IV) acetate (529,559) was purchased from Sigma-Aldrich (Shanghai) Trading Co. (Darmstadt, Germany). 1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) (LP-R4-117) was purchased from Xi’an Ruixi biological Technology Co. (Xi’an, China). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (F20170000595) was purchased from AVT (Shanghai) Pharmaceutical Technology Co. (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) (C11995500BT), DMEM/F-12 (C11330500BT), trypsin (25,200-072) and Penicillin Streptomycin (15140122) were obtained from Gibco (Grand Island, NY, United States). Fetal bovine serum (FBS) (FSP500) was purchased from Excell Bio (Jiangsu, China). Trizol lysis buffer (15596018) was obtained from Invitrogen Co. (California, United States). Phosphate buffered saline (PBS) (G0002-15), Hematoxylin and eosin (H&E) staining solution (G1076-500ML) and Masson’s trichrome staining solution (G1006) were purchased from Wuhan Servicebio Technology Co. (Wuhan, China). RIPA Lysis Buffer, reactive oxygen species assay kit (S0033S), cell counting kit-8 (C0038) and Nuclear and cytoplasmic protein extraction kit (P0028) were obtained from Beyotime Biotechnology (Shanghai, China). PolyJet™ in vitro DNA Transfection Reagent (SL100688) was purchased from SignaGen Laboratories (Maryland, United States). In Situ Cell Death Detection Kit (12156792910) was obtained from Roche Diagnostics Deutschland GmbH (Mannheim, Germany). Pierce™ BCA Protein Assay Kits (23,227), SuperSignal West Pico PLUS (34,580) and DNase I (18068015) were obtained from Thermo Fisher Scientific (Jiangsu, China). Dihydroethidium (DHE) (KGAF019, KeyGEN) was purchased from KeyGEN BioTECH (Nanjing, China). EndoFree Mini Plasmid Kit II (DP118-02) was purchased from TIANGEN Biotech Co. (Beijing, China). Anti-Nrf2 (E3J1V for WB), anti-GAPDH (14C10) and Cleaved-Caspase-3 antibody (9964S) were purchased from Cell Signaling Technology (Danvers, MA, United States). Anti-Nrf2 (ab31163 for Immunofluorescence), anti-Heme Oxygenase 1 antibody (ab68477) were obtained from Abcam (Cambridge, MA). Beta-tubulin (TA-10) was purchased from ZSGB-BIO (Wuxi, China). Lamin B1 Polyclonal Antibody (AP6001) was obtained from Bioworld Technology, Inc. (Shanghai, China). iNOS Polyclonal antibody (18985-1-AP) and HRP-conjugated secondary mouse or rabbit antibodies were obtained from Proteintech Group, Inc. (Chicago, United States). Anti-CD68 Antibody (137,001) was obtained from BioLegend (San Diego, CA). α-actinin (BM003) was obtained from Boster Biological Technology co. (California, United States). 4′,6-diamidino-2-phenylindole (DAPI) (C0065) was purchased from Beijing Solarbio Science & Technology Co. (Beijing, China). Fluorescent secondary antibody was obtained from Abbkine Biotechnology Co. (Wuhan, China). Sangon Biotech (Shanghai, China).

Briefly, oleamine (3.2 g) and cerium (IV) acetate hydrate (1.36 mM) were dissolved in the xylenes (15 mL) and stirred overnight at room temperature. Subsequently, the mixture was heated to 90°C and ddH₂O (1 mL) was added into the mixture under vigorous stirring. The mixture was further aged was aged at 90°C for 3 hours. After cooling to room temperature, the ceria nanoparticles were precipitated by adding equal volume of ethanol and harvested by centrifugation. After washing with ethanol three times, the ceria materials were dispersed in ethanol for further use.

The X-ray diffraction (XRD, 40 kV, 40 mA, D8 advanced, Bruker, Germany) and X-ray photoelectron spectrum (XPS) were employed to analyze the element of the ceria material. The Al X-ray source was used in XPS, the tube voltage was 15 kV and the tube current was 12 mA, the diameter beam spot was 500 µm (Escalab 250xi, Thermo Scientific, United States).

To prepare the positive charged ceria nanoparticles, the ceria materials (6.0 mg), the DOTAP (30.0 mg) and DOPE (30 mg) Pharmaceutical Technology Co., Shanghai, China) were dispersed in the methanol (5 mL). And then, rotary evaporation was used to completely remove the solvent of this mixture. Subsequently, the materials were re-dissolved by deionized water (20 mL) and handled by high-pressure homogenization at 800 bar for 10 min. The positive charged ceria nanoparticles were obtained following dialysis in the deionized water. The morphology of the ceria nanoparticles was imaged by the transmission electron microscope (TEM), the voltage was 100 kV (JEM 2100F, JEOL, Japan).

The plasmid encoding Nrf2 was added into the ceria nanoparticle solution with a series of nanoparticle to plasmid ratios (μL/μg) (N/P ratios, 4, 5, 7, 10, 12, 15, 17, 20, 24, 30, 40, 60, 120) and incubated at room temperature for 0.5 h. The plasmid encoding Nrf2 was successfully wrapped into the ceria nanoparticles and the plasmid - loading ceria nanoparticles complex was formed. The size and the zeta potential of the CeO₂/Nrf2 nanocomposites were analyzed by the dynamic light scattering (DLS) method (ZetaSizer, Malvern, United Kingdom). The agarose gel electrophoresis was employed to confirm the complexation. The naked plasmid and the complex with different N/P ratios were loaded, the voltage was 100 kV. The CeO₂/Nrf2 nanocomposites with N/P ratio of 5 was stored in PBS (with 10% serum) at room temperature for up to 7 days, then its stability was assessed in the aspects of particle size.

It was found that Ceria nanoparticles increased superoxide dismutase (SOD) and catalase activity. And this mimetic activity that has been assumed to be accountable for cellular defense by nanoceria. The effects of the ceria nanoparticle on the SOD activity were measured by commercial kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to instruction for authors. The ceria nanoparticles were used at different concentrations (0, 0.1, 0.2, 0.4, 1.0, 2.0, 4.0 µM).

The effects of the ceria nanoparticle on the catalase activity were further assayed by detecting the residual H₂O₂ amount. Briefly, different concentrations of the ceria nanoparticles (0, 15, 30, 60, 90, 120, 150 µM) were incubated with H₂O₂ (40 µM) at room temperature for 3 h. And then the horseradish peroxidase (HRP) and the TMB solution was further added into the mixture and incubated at 37°C for another 0.5 h. Sulfuric acid was used to terminate the reaction and the absorbance was measured at 450 nm (Epoch, Biotek, United States). Compared with the H₂O₂ standard curve, the residual H₂O₂ amount could be determined.

To investigate the targetability of the CeO₂/Nrf2 nanocomposites in vivo, we prepared DiR-abeled CeO₂ nanoparticles and CeO₂/Nrf2 nanocomposites for tracking study. The prepared nanoparticles were injected intravenously through the tail vein of C57BL/J6 mice. At 0.5–72 h after injection, images were taken using the IVIS Lumina imaging system. After in vivo tracking, the tissues (heart, lung, liver, kidneys, and spleen) of the mice were collected and subjected to ex vivo imaging (Liang et al., 2022).

In this study, all animal experiments were approved by the Animal Research Committee, Guangzhou Medical University (Guangzhou, China). All experiments were approved by the Institutional Animal Care and Use Committee, Guangzhou Medical University, Guangzhou, China (the approved ethic No. was 2019-590). Male C57BL/6J mice (9 weeks old, 20–25 g) were purchased from the Medical Experimental Animal Center of Guangdong Province and housed in conditions of temperature (23°C ± 2°C), humidity (60% ± 5%) and 12 h light-dark cycle at the Center of Laboratory Animal, Guangzhou Medical University. They were received humane care and free to food and water. All the experimental procedures were carried out according to the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 2011).

Animal model of myocardial infarction was established through ligation of the left anterior descending artery (LAD) as we previously described (Zheng et al., 2020). Briefly, the mice were anesthetized by intraperitoneally administration with 4.5% pentobarbital hydrochloride (Merial, Hallbergmoos, Germany) and connected to a respirator for mechanical ventilation. After thoracotomy between the third and the fourth rib on the left, the heart was exposed and the LAD coronary artery was ligated by using 8–0 silk suture (Ningbo Medical Needle Co., Ningbo, China). Animals in the sham group underwent the same procedure but without ligation.

Total amount of 79 mice were randomized separated into 5 different groups: (1) the sham group (sham, n = 15); (2) myocardial infarction group (MI, n = 15); (3) MI + Nrf2 plasmid alone treated group (MI + Nrf2, n = 15); (4) MI + CeO₂ nanoparticles alone treated group (MI + CeO₂, n = 17); (5) MI + the complex of CeO₂ nanoparticles with Nrf2 plasmid treated group (MI + CeO₂/Nrf2 nanocomposites, n = 17). The equivalent volume of normal saline was used as the control solvent. The Nrf2 plasmid solvent (5 ug/10 g body weight), CeO₂ nanoparticles (N/P 8:1) and the CeO₂ nanoparticles with Nrf2 plasmid (5 ug/10 g body weight, N/P 8:1) were administrated separately to mice via tail vein at different time point (0, 24 h, 3 days, 5 days or 7 days) post MI surgery. Echocardiography was performed at the third day or the 14th day post MI.

As we previously described (Wu et al., 2022), transthoracic echocardiography was performed by using Vevo 2100 imaging system (Visual Sonics, Toronto, Canada) with high-frequency ultrasonography while the mice were awake. The left ventricle was viewed at short axis M-mode. The echocardiographic parameters of left ventricle such as left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diastolic diameter (LVIDD), left ventricular internal systolic diameter (LVIDS), left ventricular end-diastolic anterior wall (LVAWD), left ventricular end-systolic anterior wall (LVAWS), left ventricular end-diastolic posterior wall (LVPWD), left ventricular end-systolic posterior wall (LVPWS) were measured.

The heart tissues were fixed with 4% paraformaldehyde overnight at 4°C. Hematoxylin and eosin (H&E) staining and Masson’s trichrome staining (Wuhan Servicebio Technology Co., Wuhan, China) were performed as we described previously (Zheng et al., 2020; Wu et al., 2018).

For immunofluorescent (IF) (Wu et al., 2022), paraffin sections of myocardial samples were treated with primary Nrf2 (1:100, Abcam, Cambridge, MA) antibody overnight at 4°C. Then, samples were incubated with secondary DyLight 594-labelled Goat Anti-Rabbit IgG (1:250, a23320; Abbkine Biotechnology Co., Wuhan, China) at room temperature for 2 h. 4′,6′-diamidino-2-phenylindole (DAPI, 10 μg/mL) was used to stain the nuclei. Fluorescence was captured by confocal microscopy and were analyzed using ImageJ software.

DHE staining was performed as we previously described (Gao et al., 2022). Briefly, the fresh heart tissues were rapidly excised, fixed in 4% paraformaldehyde and embedded in OCT Compound (TISSUE-TEK; Sakura Finetek United States, Inc.). And then, 10 μm thick sections were cut and incubated with 10 mM DHE (KGAF019, KeyGEN) at 37°C for 30 min 4′,6′-diamidino-2-phenylindole (DAPI, 10 μg/mL) was used to indicate the nuclei. Images were obtained by fluorescent microscopy and were analyzed by ImageJ software.

TUNEL staining was preformed as we previously described (Zheng et al., 2020). Briefly, myocardial apoptosis was measured with In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) according to manufacturer’s instruction. Images were attained by fluorescent microscope with 200x magnification and evaluated by ImageJ. The TUNEL positive stained cells were counted in 4 views in AAR and normalized to total area of the image determined by ImageJ.

The H9C2 cardiac myoblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in high-glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (Excell Bio, Jiangsu, China) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, United States) in an incubator containing 95% air and 5% CO₂ at 37°C. Passage 3–9 of cells was used.

To mimic myocardial ischemia in vitro, H9C2 cardiac myoblasts were subjected to oxygen-glucose deprivation (OGD) as we previously described (Wu et al., 2018). The hypoxic/ischemia group was prepared by replacing the growth medium of the cells with serum-free dulbecco’s modified eagle medium nutrient mixture F-12(Ham) (Gibco, Grand Island, NY, United States), and then incubating the H9C2 cardiac myoblasts at 37°C in an incubator containing 94% N₂, 5% CO₂, and 1% O₂ for 6 h. Control cells were kept in normal medium under normal conditions. To examine the effect of CeO₂ nanomaterials and CeO₂/Nrf2 nanocomposites on OGD-induced cardiomyocyte injury, H9C2 cardiac myoblasts were treated with or without CeO₂ nanomaterials for 18 h followed by OGD for 6 h before the next experiments. all the experiments were repeated 3 times.

RAW264.7 macrophages were obtained from American Type Culture Collection (ATCC, Rockville, MD), and cultured in high-glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Excell Bio) and 1% penicillin/streptomycin in an incubator containing 95% air and 5% CO₂ at 37°C.

For DNA transfection (Chuang et al., 2014), the NFE2L2 plasmid (NM_006164) (Beijing Tsingke Biotech Co.) were transfected into H9C2 cardiac myoblasts according to the manufacturer’s instructions. Cells were plated 24 h prior to transfection. For each well of a 6-well plate, 2 µg of DNA was diluted into 100 µL of serum-free DMEM with high glucose. Additionally, 6 µL of PolyJet™ reagent (SignaGen Laboratories, Maryland United States) was diluted into 100 µL of serum-free DMEM with high glucose. The diluted PolyJet™ reagent was immediately added to the diluted DNA solution all at once. The solution was vortex-mixed and incubated for 10–15 min at room temperature to allow transfection complexes to form. At the end of incubation, 2.0 mL of pre-warmed complete cell growth medium was added to the cells and plated onto a well of a 6-well plate. The cells were incubated at 37°C with 5% CO₂. The transfection complex-containing medium was gently removed and replaced with complete culture medium 12–16 h after plating. Transfection efficiency was checked 48 h post-transfection.

H9C2 cardiac myoblasts were seeded in 96-well plates at 6 × 10³ cells per well and incubated for 24 h. Subsequently, the cells were treated with varying concentrations of CeO₂ nanoparticles. As we have described previously (Gao et al., 2022), Cell viability was assessed using the CCK-8 assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Briefly, 10 µL of CCK-8 reagent was added to each well containing culture medium, followed by incubation at 37°C in the dark for 1 h. Absorbance of each well was measured at 450 nm using a spectrophotometer. Furthermore, to evaluate cell viability of the different treatment groups, the CCK-8 assay was conducted again after hypoxia exposure.

Intracellular ROS levels were assessed using DCFH-DA probe (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, the cells were treated with DCFH-DA (10 μM) for 20 min at 37°C. After incubation, the cells were washed for 3 times and then Medium was added for microscopic imaging.

Images were acquired with Nikon A1 (Nikon America Inc., Melville, NY) confocal microscope and the intensity was measured by ImageJ software. For quantification, 3 images of different views were taken blindly and the green fluorescence intensity was counted by ImageJ. Representative images/figures were selected according to their quality and to most accurately represent the group mean/average across all the available data.

As we described previously (Zheng et al., 2020), the tissue of the infarct hearts was separated and homogenized, the nucleus and cytoplasmic fractions of treated cells were collected by a Nuclear and Cytoplasmic Extraction Reagents kit (Beyotime, Shanghai, China). The protein concentration was determined using the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Jiangsu, China). Protein samples were separated by electrophoresis using a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, United States). The PVDF membrane was blocked and incubated with the primary antibody overnight at 4°C. And then, the membrane was further incubated with secondary antibody for 1 h at room temperature. Blots were developed using the SuperSignal West Pico PLUS (Thermo Fisher Scientific, Jiangsu, China). The band intensity was analyzed with ImageJ software.

Myocardial tissues or H9C2 cardiac myoblasts were homogenized in TRIzol (1 mL, Invitrogen, California, United States) to extract total RNA. The cDNA was amplified by using AG RNAex Pro Reagent (Accurate Biology, Hunan, China) and qPCR was performed as we described previously (Zheng et al., 2020). Primer sequences were listed in Supplementary material online (Supplementary Table S1).

All data was analyzed with GraphPad Prism, version 8.0 (GraphPad Software, Boston, MA, United States) and shown as means ± SEM. Difference between two groups were analyzed using Student’s t-test. For three or more groups, the normal distribution was confirmed by the Shapiro–Wilk test (P > 0.1) with SPSS 17.0. Differences among three or more groups were analysed using one-way ANOVA. Statistical significance was accepted for P < 0.05. Further Tukey post hoc analysis (α = 0.05) was performed to confirm where the differences occurred between groups. The detailed P-value compared with respective controls is presented in the corresponding Figure.

Ceria nanoparticles were synthesized using a previously published simple wet chemistry method (Li et al., 2022). The morphology of the ceria nanoparticles was observed using transmission electron microscope (TEM). As shown in Figure 1A, the ceria nanoparticles were well-dispersed without significant agglomeration, with an average diameter of approximately 30 nm. Further analysis of the size and zeta potential of the ceria nanoparticles was conducted. Dynamic light scattering (DLS) results showed an average nanoparticle size of 326.8 ± 138.9 nm (PDI: 0.271) and a zeta potential of 45.2 ± 9.9 mV (Figures 1B, C).

After loading with Nrf2 plasmids, the nanoparticle size decreased to approximately 190.7 ± 80.3 nm, and the zeta potential decreased with increasing nucleic acid ratios. Specifically, the zeta potential declined to 15.4 ± 6.2 mV at an N/P ratio of 5. The CeO₂/Nrf2 nanoparticles were negatively charged at an N/P ratio of 4, indicating plasmid overload. The reduction in particle size and positive zeta potential confirmed the successful condensation of the ceria nanoparticles with the nucleic acid, as further validated by agarose gel electrophoresis (Figure 1D). The optimized N/P ratio of 5 indicated that the ceria nanoparticles had a promising nucleic acid loading capacity. In addition, the particle size measurements indicated that the CeO₂/Nrf2 nanocomposites had good stability (Figure 1I).

Subsequently, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were employed to determine the valence states of cerium in the nanoparticles. In the XRD spectrum, the (002) peak was attributed to Ce³⁺, while the (111), (220), and (311) peaks were attributed to Ce⁴⁺ (Figure 1E). The Ce 3 days spectrum was deconvoluted into two groups of peaks: 883.80, 891.15, 900.75, 903.00, 912.30, 918.75 eV corresponding to Ce⁴⁺, and 886.40, 907.40 eV corresponding to Ce³⁺. The results indicated that both Ce³⁺ and Ce⁴⁺ co-exist in the ceria nanoparticles, with a ratio of approximately 1:1.9 (Ce³⁺/Ce⁴⁺) (Figure 1F).

The transition between Ce³⁺ and Ce⁴⁺ is believed to facilitate the elimination of ROS. To comprehensively assess the antioxidant capacity of the synthesized cerium nanoparticles, free radical scavenging experiments were conducted at varying concentrations. The superoxide dismutase (SOD) and catalase mimetic activities of the ceria nanoparticles were measured. The ceria nanoparticles were capable of eliminating superoxide anions and reducing absorbance at 560 nm. As shown in Figure 1G, the SOD mimetic activity of the ceria nanoparticles increased proportionally with the concentration of ceria. The linear relationship between the 1/inhibition rate and the 1/ceria concentration further validated this observation. Similarly, the ceria nanoparticles effectively eliminated H₂O₂ in a concentration-dependent manner as demonstrated in Figure 1H. Additionally, the cytotoxicity of the ceria nanoparticles was assessed using H9C2 cardiac myoblasts. As shown in Supplementary Figure S1A, CeO₂ nanoparticles exhibited no significant cytotoxicity in H9C2 cardiac myoblasts. These findings indicate that the ceria nanoparticles and CeO₂/Nrf2 nanocomposites possess SOD and catalase mimetic activities without cytotoxic effects.

Efficient cellular internalization is essential for effective gene transfection (Wang et al., 2018). Initially, Nrf2 plasmids were successfully transfected and expressed in H9C2 cardiac myoblasts using Lipofectamine 2000 (Supplementary Figure S2A). Subsequently, CeO₂/Nrf2 nanocomposites were prepared at various N/P ratios and incubated with H9C2 cardiac myoblasts. As shown in Figures 2A, B, Nrf2 expression was significantly elevated, reaching a peak at an N/P ratio of 8, indicating that CeO₂ nanomaterials can efficiently transfect Nrf2 plasmids into cardiomyocytes. The optimal N/P ratio for CeO₂/Nrf2 nanocomposites was determined to be 8. Furthermore, the successful uptake of the Nrf2 plasmid was further confirmed by labeling the plasmid with the fluorescent dye FAM. Macrophages were incubated with CeO₂/FAM-labeled Nrf2 nanocomposites for different durations (0 h, 0.5 h, 2 h, and 4 h). As shown in Figure 2C, the CeO₂/Nrf2 nanocomposites were rapidly internalized by macrophages within 0.5 h and localized around the nucleus.

To monitor the distribution and targeting ability of the CeO₂/Nrf2 nanocomposites to the cardiac infarction zone, a mouse model was established via permanent ligation of the left anterior descending artery. The CeO₂/Nrf2 nanocomposites or CeO₂ nanomaterials were administered to mice via tail vein injection within 0.5-to-1-h post-MI. As shown in Figures 2D, E, both CeO₂ nanomaterials and CeO₂/Nrf2 nanocomposites distributed throughout the entire body within 72 h after injection, with a strong signal observed in the heart. At 24- and 72-h post-injection, the organs (heart, lung, liver, kidneys, and spleen) were isolated. Significant accumulation of CeO₂ nanomaterials and CeO₂/Nrf2 nanocomposites in the heart was observed after 24 h (Figures 2F, G). This CeO₂/Nrf2 nanocomposite-based delivery system, administered via tail vein injection, demonstrated effective accumulation in the heart. Distribution in other organs was also detected (Figure 2H). Moreover, H&E staining results showed that CeO₂/Nrf2 nanocomposites did not alter the histological features of these organs (Supplementary Figure S2B). These findings indicate that cerium oxide nanomaterials facilitate the accumulation and retention of Nrf2 in the heart with myocardial infarction.

The objective of this study was to deliver Nrf2, a key regulator during oxidative stress, to the heart using ceria nanoparticles. To validate the in vivo delivery efficacy of ceria nanoparticles for Nrf2, we prepared Nrf2 plasmid, CeO₂ nanoparticles, and CeO₂/Nrf2 nanocomposites, which were administered to mice via tail vein injection within 0.5–1 h post-MI surgery, with repeated doses on the first and third days post-MI (Figure 3A). Compared to the groups treated with Nrf2 plasmid or CeO₂ nanoparticles alone, the mRNA and protein levels of Nrf2 were significantly higher following treatment with CeO₂/Nrf2 nanocomposites (Figures 3B–D). Additionally, immunofluorescence results indicated that the red fluorescence intensity of Nrf2 was stronger in the CeO₂/Nrf2 nanocomposite-treated group compared to the groups treated with Nrf2 plasmid alone or ceria nanoparticles alone (Figures 3E, F). These results consistently demonstrated that CeO₂/Nrf2 nanocomposites effectively delivered Nrf2 into the heart tissue in a myocardial infarction model.

CeO₂/Nrf2 nanocomposites, along with Nrf2 plasmid and CeO₂ nanomaterials, were prepared and administered to mice post MI via tail vein injection within 0.5–1 h post-MI surgery. The administrations were repeated on days 1, 3, 5, and 7. Cardiac function was assessed using transthoracic echocardiography on days 3 and 14 post-MI (Supplementary Figure S3A; Supplementary Table S2). As expected, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were significantly reduced, while left ventricular internal diameter (LVID) was increased at days 3 and 14 post-MI (Figure 4A; Supplementary Figure S3B; Supplementary Table S2). Treatment with CeO₂ nanoparticles alone or CeO₂/Nrf2 nanocomposites slightly improved these parameters. The CeO₂/Nrf2 nanocomposites group exhibited superior cardioprotective effects compared to the Nrf2 plasmid or CeO₂ nanoparticles alone groups. Masson trichrome staining was used to analyze cardiac fibrosis and collagen deposition post-MI. The results showed that fibrosis and collagen deposition were significantly alleviated in the CeO₂/Nrf2 nanocomposite-treated mice compared to those treated with vehicle, Nrf2 plasmid alone, or CeO₂ nanoparticles alone (Figures 4B, C). In summary, these findings suggest that CeO₂/Nrf2 nanocomposites protect against MI-induced cardiac dysfunction and pathological remodeling in mice.

Apoptotic cell death of cardiomyocytes is a critical mechanism contributing to irreversible damage in MI (Chiong et al., 2011; Sheng et al., 2023). The protein level of Cleaved caspase-3, a key apoptosis effector, increased post-MI and was reduced by treatment with CeO₂/Nrf2 nanocomposites or CeO₂ nanoparticles alone (Figures 4D, E). Compared to the sham group, there was a significant increase in TUNEL-positive cells in the MI group, which was significantly reduced by treatment with CeO₂/Nrf2 nanocomposites or CeO₂ nanoparticles alone (Figures 4F, G). Additionally, CeO₂/Nrf2 nanocomposites demonstrated a stronger protective effect against cardiomyocyte apoptosis than CeO₂ nanoparticles alone.

The surge of oxidative stress and inflammatory responses in the early stages post MI plays a crucial role in the progression of ischemic myocardial injury (Xiang et al., 2021). Nrf2, a dominant transcriptional factor in cellular defense against oxidative and xenobiotic stresses, activates downstream target genes such as heme oxygenase 1 (HO-1, also known as HMOX1) and NAD(P)H dehydrogenase quinone 1 (NQO1) (Wu et al., 2022; Mohs et al., 2021). To explore the protective mechanism of CeO₂/Nrf2 nanocomposites, the level of intracellular ROS accumulation in the infarcted area was measured. DHE assays showed a burst of ROS production in the heart 3 days post-MI (Figures 5A, B). While treatments with Nrf2 plasmid and CeO₂ nanoparticles alleviated ROS production, CeO₂/Nrf2 nanocomposites exhibited stronger effects (Figures 5A, B). Furthermore, the mRNA levels of HO-1 and NQO1 were significantly elevated with CeO₂/Nrf2 nanocomposites treatment in mice 3 days post-MI (Figure 5C), confirming the activation of Nrf2 defense mechanisms.

The interaction between oxidative stress and inflammation contributes to the progression and prognosis of MI (Wang et al., 2018). Therefore, the effects of CeO₂/Nrf2 nanocomposites on inflammatory responses post-MI were further investigated. Immunohistochemical staining showed that CD68 (+) macrophages infiltrated the infarcted area 3 days post-MI, which was significantly reduced following CeO₂/Nrf2 nanocomposites treatment (Figures 5D, E). The fluorescence intensity of iNOS, a marker of M1 macrophages, positively correlated with CD68 (+) indicated that CeO₂/Nrf2 nanoparticles reduced M1 macrophages, suggesting their role in mitigating acute inflammatory responses. On the other side, there is no significant difference on M2 macrophages in the heart of different groups as evidence by co-localization of CD68 and Arg1 (a marker for M2 macrophages) (Supplementary Figure S4). Moreover, the mRNA levels of pro-inflammatory genes were elevated, and those of anti-inflammatory factors (TGFβ1, IL-6, and TNFα) were reduced 3 days post-MI. CeO₂/Nrf2 nanocomposites treatment significantly ameliorated these MI-induced abnormal gene expression changes (Figure 5F). These results strongly indicate the antioxidant and anti-inflammatory properties of CeO₂/Nrf2 nanocomposites during MI.

To further elucidate the protective effects of CeO₂/Nrf2 nanocomposites in cardiomyocytes, H9C2 cardiac myoblasts were used and subjected to oxygen-glucose deprivation (OGD) stimulation to mimic in vivo myocardial ischemia. The protein expressions of Nrf2, in both the nuclei and the cytoplasm, were significantly increased by CeO₂/Nrf2 nanocomposites, indicating effective delivery of Nrf2 plasmid into cardiomyocytes under OGD stimulation for 6 h (Figures 6A, B). Cell viability assays showed that CeO₂/Nrf2 nanocomposites (at different N/P ratios) significantly improved the viability of cardiomyocytes under OGD stimulation (Figure 6C). Furthermore, the incubation of CeO₂/Nrf2 nanocomposites with cardiomyocytes under OGD conditions at different N/P ratios significantly increased the protein levels of Nrf2 and HO-1, while decreasing the protein level of Cleaved-caspase3 (Figures 6D, E). Consistent with our previous results, the optimal N/P ratio for CeO₂/Nrf2 nanocomposites was determined to be 8.

The DCFH-DA staining results indicated that oxidative stress levels in H9C2 cardiac myoblasts were elevated following OGD stimulation. Compared to the groups treated with either Nrf2 plasmid transfection or ceria nanoparticles alone, CeO₂/Nrf2 nanocomposites exhibited a stronger effect in reducing ROS production in cardiomyocytes under OGD stimulation (Figures 6F, G). Consistent with our in vivo findings, CeO₂/Nrf2 nanocomposites effectively protected cardiomyocytes from injury by elevating Nrf2 expression.