Abstract Ferroptosis plays a critical role in myocardial ischemia-reperfusion injury (MIRI), posing a significant clinical challenge. Nanoenzymes like cerium oxide (CeO[2]) hold promise for mitigating oxidative damage and inhibiting ferroptosis, but their delivery efficiency and biological activity require optimization. This study aims to develop a targeted nanozyme delivery system for MIRI treatment by integrating CeO[2] with mesoporous polydopamine (mPDA) and dexrazoxane (DXZ) to achieve synergistic therapeutic effects. A biomineralization technique was used to synthesize CeO[2] nanoparticles (2–3 nm) within mPDA, forming ~ 130 nm composite nanoparticles (Ce@mPDA). Surface modifications with cardiac homing peptide (CHP) and triphenylphosphine (TPP) enabled hierarchical targeting to injured myocardium and mitochondria. DXZ-loaded Ce@mPDA-C/P nanoparticles (D/Ce@mPDA-C/P) were evaluated in vitro and in a MIRI mouse model for their effects on oxidative stress, ferroptosis, apoptosis, inflammation, and cardiac function. D/Ce@mPDA-C/P nanoparticles exhibited robust ROS scavenging, sustained DXZ release, and efficient myocardial and mitochondrial targeting. The D/Ce@mPDA-C/P system significantly reduced oxidative stress, upregulated GPX4 expression, inhibited ferroptosis, and modulated the inflammatory microenvironment. Long-term studies in a MIRI mouse model demonstrated reductions in myocardial fibrosis and improvements in cardiac function, including enhanced fractional shortening and ejection fraction. This hierarchical targeting delivery system effectively combines the antioxidant properties of CeO[2] with the iron-chelating effects of DXZ, providing a promising therapeutic strategy for MIRI. This approach may expand the clinical use of DXZ and advance nanomedicine-based interventions for myocardial repair. Graphical Abstract [38]graphic file with name 12951_2025_3197_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03197-1. Keywords: Myocardial ischemia-reperfusion injury, CeO[2] nanozymes, Mesoporous polydopamine, Dexrazoxane, Mitochondria Introduction With the aging society and changing lifestyles, the incidence of cardiovascular diseases is continuously increasing, posing significant pressure on global public health. Myocardial infarction (MI) is one of the leading causes of cardiovascular disease-related deaths worldwide [[39]1]. Currently, the primary treatment for acute myocardial infarction is percutaneous coronary intervention (PCI), which effectively restores blood flow, reduces myocardial ischemia, and improves clinical prognosis [[40]2]. However, despite the significant number of myocardial cells saved by PCI, it is also associated with severe complications myocardial ischemia-reperfusion injury (MIRI), which often results in further myocardial injury and even worsens heart failure [[41]3]. A key pathological mechanism in MIRI is ferroptosis—an iron-dependent form of cell death driven by lipid peroxidation [[42]4, [43]5]. Unlike traditional apoptosis or necrosis, ferroptosis is primarily characterized by the accumulation of lipid peroxides and dysregulated iron metabolism, with excessive production of reactive oxygen species (ROS) serving as a critical contributing factor. In the field of nanomedicine, nanoenzymes (such as CeO[2]) have garnered significant attention due to their excellent antioxidant properties. Cerium oxide (CeO[2]), as an enzyme-like substance, can efficiently scavenge ROS inside cells through its redox properties, thereby reducing oxidative stress and cellular damage [[44]6, [45]7]. Compared to traditional antioxidant strategies (such as natural antioxidants), CeO[2] nanoenzymes have distinct advantages: CeO[2] can generate and consume ROS through a rapid redox cycle, exhibiting strong stability and reusability [[46]8]. However, the particle size of CeO[2] significantly impacts its biological activity and delivery efficiency. Smaller-sized CeO[2] nanoparticles have higher catalytic activity, as smaller particles provide more surface-active sites, and CeO[2] particles in the range of 2–20 nm are commonly used in biomedical applications [[47]9–[48]11]. However, it is not common to use small-sized CeO[2] nanoparticles alone to treat myocardial ischemia-reperfusion injury because their smaller size limits their delivery efficiency in vivo. Studies have demonstrated that nanoparticles sized between 20 and 200 nm are highly efficient for intravenous delivery, as their dimensions allow them to pass through intercellular gaps in myocardial vascular endothelial cells induced by MIRI while minimizing liver accumulation, thereby facilitating targeted delivery to myocardial tissue [[49]12–[50]14]. Polydopamine (PDA), a melanin-like material, is known for its excellent biocompatibility and effective nanoparticle modification capabilities [[51]15]. In this study, we employ an innovative approach using mesoporous PDA (mPDA) as a carrier material. Through biomineralization, we grow 2–3 nm CeO[2] nanoparticles in situ on mPDA, forming a composite nanoparticle of approximately 100 nm in size, which addresses the issue of low delivery efficiency seen with small-sized CeO[2] particles while avoiding the poor activity of larger CeO[2] particles [[52]9–[53]11]. CeO₂, renowned for its catalase (CAT)-like and superoxide dismutase (SOD)-like activities, efficiently scavenges H[2]O[2] and O[2]⁻ but shows relatively limited activity against •OH [[54]9–[55]11]. Notably, CeO[2] exhibits peroxidase (POD)-like activity, this property can, under certain conditions, promote the generation of •OH through Fenton-like reactions involving H[2]O[2]. This paradoxical effect is one of the reasons why CeO[2] alone shows limited efficacy in scavenging •OH radicals, which are the most damaging ROS species and a key contributor to MIRI [[56]16]. Based on this, we utilized the unique mesoporous structure of mPDA to load dexrazoxane (DXZ), an iron chelator, has shown significant therapeutic effects on MIRI in preclinical studies. DXZ can chelate excess Fe^2+ in cardiomyocytes, thereby reducing the Fenton reaction and the subsequent production of •OH [[57]17, [58]18]. By combining CeO[2] with DXZ, this nanozyme system can comprehensively target multiple types of ROS. Specifically, CeO[2] scavenges upstream ROS, such as H[2]O[2] and O[2]^−, while DXZ effectively inhibits the generation of downstream •OH, ensuring a more complete ROS clearance. This synergistic approach leverages the strengths of both components, providing a robust strategy for mitigating oxidative stress in MIRI and potentially overcoming the limitations of standalone antioxidant therapies. Importantly, mitochondria, as the cell’s primary energy factory and iron metabolism center, play a pivotal role in ferroptosis. Mitochondrial dysfunction not only leads to metabolic disturbances but also exacerbates ROS generation, further promoting lipid peroxidation and cell damage [[59]19]. We modified the surface of mPDA with cardiac homing peptide (CHP) and triphenylphosphine (TPP). The CHP enables the nanoparticles to specifically target myocardial injury sites [[60]20], while TPP facilitates the delivery of the drug to mitochondria [[61]21]. This hierarchical targeting strategy allows the mPDA composite to effectively cross biological barriers, target damaged myocardium, and enter myocardial cell mitochondria, thus achieving more precise therapeutic effects. Results Synthesis and characterization of CeO[2] Nanozyme-Mesoporous polydopamine nanoparticles We first synthesized CeO[2]-loaded mesoporous polydopamine (Ce@mPDA) nanoparticles and all Ce@mPDA nanoparticles used in the experiments were PEG-modified to achieve better stability [[62]22]. Ce@mPDA nanoparticles intended for targeted delivery to injured myocardium were modified with NH[2]-PEG[2000]-CHP (Ce@mPDA-C), while those designed for mitochondrial delivery in injured myocardium were co-modified with NH[2]-PEG[2000]-CHP and NH[2]-PEG[2000]-TPP (Ce@mPDA-C/P). The transmission electron microscopy (TEM) image (Fig. [63]1A) reveals the clear mesoporous structure of the Ce@mPDA-C/P nanoparticles. At higher magnification, the CeO[2] lattice can be observed, confirming the successful loading of CeO[2] onto mPDA. Elemental mapping further verifies the presence of Ce and P elements on the nanoparticle surface, confirming both CeO[2] loading and TPP modification. X-ray photoelectron spectroscopy (XPS) analysis (Fig. [64]1B) shows the coexistence of Ce(III) and Ce(IV) oxidation states on the Ce@mPDA-C/P surface. This dual valency is essential for CeO[2]’s ability to cyclically scavenge ROS, indicating its potential to mitigate oxidative stress in cells. Dynamic light scattering (DLS) analysis (Fig. [65]1C) reveals that the particle size of Ce@mPDA-C/P nanoparticles is between 115 and 150 nm, with a peak around 135 nm. This size range is optimal for intravenous injection, ensuring efficient delivery to the damaged myocardial tissue [[66]12]. Fig. 1. [67]Fig. 1 [68]Open in a new tab Characterization and functionality evaluation of Ce@mPDA-based nanoparticles. (A) TEM images and elemental mapping of Ce@mPDA-C/P nanoparticles, showing the morphology, core-shell structure, and elemental distribution. High-resolution image indicating lattice spacing of 2.9 Å. (B) XPS spectra of Ce in Ce@mPDA-C/P nanoparticles. (C) Particle size distribution of Ce@mPDA-C/P nanoparticles. (D) ROS scavenging activities of various nanoparticles (mPDA, Ce@mPDA, Ce@mPDA-C, and Ce@mPDA-C/P) against, H[2]O[2], and O[2]⁻. (E) •OH scavenging activity of Ce@mPDA-C/P nanoparticles with and without DXZ. (F) Drug (DXZ) loading efficiencies of different nanoparticles. (G) Hydrodynamic size and (H) Zeta potential measurements of different nanoparticles. (I) Stability test of Ce@mPDA-C/P nanoparticles over 7 days, including size and polydispersity index (PDI). (J) In vitro cumulative release profile of DXZ from Ce@mPDA-C/P nanoparticles. K) In vivo fluorescence imaging of myocardial tissue in MIRI mice 4 h post-injection. L) Quantitative analysis of fluorescence intensity of ICG-loaded Ce@mPDA-based nanoparticles and free ICG. Data are presented as the mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns ≥ 0.05 We further assessed the ROS scavenging efficiency of various nanoparticles. As shown in Fig. [69]1D, CeO[2]-loaded nanoparticles (Ce@mPDA, Ce@mPDA-C, and Ce@mPDA-C/P) demonstrated significantly higher ROS scavenging capacity compared to the mPDA nanoparticles that did not contain CeO[2]. Surface modifications with TPP and CHP did not significantly enhance the ROS scavenging performance, suggesting that CeO[2] loading is the main contributor to the antioxidant properties of these nanoparticles, while TPP and CHP primarily influence the delivery characteristics without greatly affecting ROS removal. Each type of ROS plays a distinct role in oxidative stress and tissue damage. H[2]O[2] and O[2]⁻ act as stable intermediates that propagate oxidative damage, while •OH, being highly reactive, directly damages cellular structures [[70]23]. In MIRI, excessive production of H[2]O[2] and over-release of Fe^2+ amplify •OH generation through the Fenton reaction, worsening oxidative stress and myocardial injury [[71]24, [72]25]. Ce@mPDA effectively scavenges H[2]O[2] and O[2]⁻ but shows limited activity against •OH (Fig. [73]1D). This limitation may be attributed to the high reactivity and short lifespan of •OH, making them more difficult to capture and remove [[74]9–[75]11]. We speculate that combining Ce@mPDA with the Fe-chelating agent DXZ can synergistically inhibit ROS production and •OH generation, providing a comprehensive strategy to alleviate oxidative stress in MIRI. Figure [76]1E demonstrates the efficiency of Ce@mPDA-C/P nanoparticles, in combination with DXZ, in scavenging hydroxyl radicals (•OH). The results show that DXZ can act synergistically with the CeO[2] nanomaterial to reduce •OH radicals generated in Fenton reactions, highlighting the potential of CeO[2] and DXZ in mitigating oxidative stress. The mesoporous structure of mPDA provides it with excellent drug-loading and sustained drug-release capabilities [[77]26]. The drug loading efficiency of DXZ on different nanoparticles is presented in Fig. [78]1F. The results indicate no significant differences in DXZ loading efficiency across the mPDA, Ce@mPDA, Ce@mPDA-C, and Ce@mPDA-C/P nanoparticles, suggesting that the modifications do not interfere with the drug-loading capacity of the mesoporous structure. In Fig. [79]1G, DLS measurements show an increase in the hydrated particle size after PEG-CHP modification, which is consistent with previously reported findings. ζ-potential measurements (Fig. [80]1H) reveal that mPDA nanoparticles possess a negative surface charge, which becomes less negative after CeO[2] loading. After TPP modification, the ζ-potential becomes positive (Ce@mPDA-C/P), which is beneficial for mitochondrial targeting. The positive charge facilitates nanoparticle interaction with the negatively charged mitochondrial membrane, enhancing the targeted delivery of nanoparticles to the mitochondria. Stability test of D/Ce@mPDA-C/P nanoparticles over 7 days, showing consistent particle size (approximately 130 nm) and a stable polydispersity index (PDI) below 0.15 (Fig. [81]1I). During this process, the loading and release of DXZ did not affect the stability of the D/Ce@mPDA-C/P nanoparticles. The drug release profile of D-Ce@mPDA-C/P nanoparticles is shown in Fig. [82]1J. These nanoparticles are capable of releasing DXZ continuously for more than 12 h, indicating their potential for sustained drug delivery. The in vivo targeted drug delivery effect in a MIRI mouse model is illustrated in Fig. [83]1K and Figure [84]S1. Fluorescently labeled nanoparticles were injected into MIRI mice, and at 4 h post-injection, major organs were imaged using fluorescence. The results show that Ce@mPDA-C and Ce@mPDA-C/P nanoparticles significantly accumulated in the damaged myocardial tissue. Quantitative analysis revealed that CHP modification was crucial for enhancing myocardial targeting, while TPP modification did not further improve the targeting effect (Fig. [85]1L). Given that D-Ce@mPDA-C/P nanoparticles can release DXZ over 10 h, we hypothesize that these nanoparticles can effectively deliver DXZ to the injured myocardium, providing prolonged drug release. In Vitro evaluation of antioxidant and ferroptosis-inhibitory effects To validate the cellular and mitochondrial targeting capabilities of the nanoparticles, we used a live-cell imaging workstation to observe the distribution of Cy7-labeled Ce@mPDA, Ce@mPDA-C, and Ce@mPDA-C/P nanoparticles in H/R-treated H9C2 cardiomyocytes. As shown in Fig. [86]2A, the overlap of Cy7 fluorescence (red) with MitoTracker fluorescence (green) indicates that the nanoparticles successfully localized to the mitochondria (Figure [87]S2). Among the tested groups, Ce@mPDA-C/P exhibited the most pronounced colocalization, with clearer fluorescence boundaries. The Cy7 fluorescence intensity of the Ce@mPDA-C group was higher than that of Ce@mPDA, suggesting that CHP modification enhances the nanoparticles’ targeting to injured H9C2 cells (Figure [88]S3). Furthermore, Ce@mPDA-C/P showed the strongest Cy7 fluorescence, potentially due to the high mitochondrial content in H9C2 cells, which increased overall nanoparticle uptake. Fig. 2. [89]Fig. 2 [90]Open in a new tab In Vitro Evaluation of Antioxidant and Ferroptosis-Inhibitory Effects. (A) Fluorescent imaging of Cy7-labeled nanoparticles (red) and mitochondria (green) using MitoTracker to assess mitochondrial targeting efficiency. (B) Quantitative analysis of MitoTracker fluorescence normalized to the control. (C) Ratio of normalized Cy7 fluorescence to normalized MitoTracker fluorescence (n = 8). (D) Flow cytometry analysis of mitochondrial ROS levels using MitoSOX staining for various treatment groups. (E) Mean fluorescence intensity (MFI) of MitoSOX from flow cytometry analysis. (F) Flow cytometry analysis of cell apoptosis using Annexin V/PI staining for different treatments. (G) Quantitative analysis of the apoptotic cell ratio based on flow cytometry results. (H) Western blot analysis of ferroptosis-related proteins (GPX4 and ACLS4) under different treatments. GAPDH was used as a loading control. (I) Quantitative analysis of ACLS4 protein and (J) GPX4 protein expression normalized to GAPDH. Data are presented as the mean ± standard deviation (n = 6, unless otherwise stated). *p < 0.05, **p < 0.01, ***p < 0.001, ns ≥ 0.05 The intensity of MitoTracker fluorescence is correlated with mitochondrial activity [[91]27]. Quantitative analysis (Fig. [92]2B) revealed that TPP modification further enhanced the mitochondrial protective effects of the nanoparticles. These results demonstrate that CHP and TPP modifications synergistically achieve hierarchical targeted delivery to injured cardiomyocytes and their mitochondria. We next calculated the ratio of normalized Cy7-labeled nanoparticle fluorescence to normalized mitochondrial fluorescence intensity across different groups (Fig. [93]2C). A higher ratio reflects a greater accumulation of nanoparticles within the active mitochondria. The results showed that Ce@mPDA-C did not significantly enhance the nanoparticle fluorescence intensity after normalization to mitochondrial fluorescence compared to Ce@mPDA. However, Ce@mPDA-C/P demonstrated a significant increase in nanoparticle fluorescence intensity after normalization to mitochondrial fluorescence compared to Ce@mPDA-C. These findings suggest that the TPP modification alone was responsible for effectively enhancing the mitochondrial targeting of the nanoparticles. To evaluate the ability of the nanoparticles to scavenge mitochondrial ROS, we used MitoSOX staining to detect mitochondrial ROS levels in H/R-treated H9C2 cells. Flow cytometry results (Fig. [94]2D) and quantitative analysis (Fig. [95]2E) showed that all nanoparticle-treated groups significantly reduced mitochondrial ROS levels. Among them, Ce modification markedly enhanced the ROS-scavenging ability of mPDA nanoparticles. CHP and TPP modifications further improved ROS clearance, while the ROS levels in the DXZ + Ce@mPDA-C/P group were even lower than those in the Ce@mPDA-C/P group, suggesting a synergistic effect between DXZ and CeO[2] nanozymes. Notably, the D-Ce@mPDA-C/P group (DXZ loaded within mPDA) exhibited the lowest ROS levels, further confirming that hierarchical mitochondrial-targeted delivery to cardiomyocytes can significantly enhance the antioxidant efficacy of DXZ. To assess the inhibitory effect of the nanoparticles on H/R-induced apoptosis in H9C2 cells, Annexin V/PI staining was performed, and the levels of apoptosis were analyzed by flow cytometry (Fig. [96]2F). Quantitative results (Fig. [97]2G) showed that H/R treatment significantly increased the apoptotic ratio, while all nanoparticle-treated groups effectively reduced apoptosis. The Ce-modified Ce@mPDA group showed better performance than the mPDA group, indicating that the antioxidative properties of CeO[2] play a crucial role in reducing H/R-induced apoptosis. Additionally, CHP and TPP modifications further enhanced the targeted delivery efficiency of the nanoparticles, with Ce@mPDA-C and Ce@mPDA-C/P groups exhibiting more pronounced anti-apoptotic effects. The DXZ + Ce@mPDA-C/P group showed a further reduction in the apoptosis rate, indicating a synergistic effect between DXZ and CeO[2] in mitigating oxidative stress-induced apoptosis. The D-Ce@mPDA-C/P group (DXZ loaded within mPDA) demonstrated the lowest apoptotic ratio, further proving that hierarchical delivery of DXZ to mitochondria significantly enhances its anti-apoptotic efficacy. To investigate the regulatory effects of the nanoparticles on ferroptosis in H/R-treated H9C2 cells, we analyzed the expression levels of ferroptosis-related proteins GPX4 (an anti-ferroptotic marker) and ACSL4 (a ferroptotic marker) (Fig. [98]2H) [[99]28]. Quantitative analysis (Fig. [100]2I, J) showed that GPX4 expression was significantly decreased, and ACSL4 expression was significantly increased in the H/R-treated group. Compared to the H/R control group, all nanoparticle-treated groups significantly increased GPX4 expression and decreased ACSL4 expression, confirming the crucial role of CeO[2] in mitigating ferroptosis. Ce@mPDA-C and Ce@mPDA-C/P groups showed more pronounced effects, indicating that CHP and TPP modifications further enhanced the ferroptosis-inhibitory effects through improved targeted delivery efficiency. In the DXZ + Ce@mPDA-C/P group, GPX4 expression was higher and ACSL4 expression was lower than those in the Ce@mPDA-C/P group, demonstrating a significant synergistic effect between DXZ and CeO[2] in alleviating H/R-induced ferroptosis. The D-Ce@mPDA-C/P group (DXZ loaded within mPDA) exhibited the best regulatory effects, confirming that hierarchical mitochondrial-targeted delivery of DXZ can further enhance its ferroptosis-inhibitory capabilities. Evaluation of Iron distribution, oxidative stress, and Cellular apoptosis in MIRI Mouse Model In the MIRI mouse model, we administered different treatments on day 4 after MIRI and collected mouse heart tissues for analysis. By evaluating iron distribution, oxidative stress levels, and cellular apoptosis, we further explored the therapeutic effects of the different nanoparticle-based treatment strategies. First, we quantified the levels of total iron, non-heme iron, and heme iron in heart tissues. The results showed no significant differences in total iron levels among the different treatment groups (Fig. [101]3A). However, the non-heme iron levels were significantly lower in the DXZ-loaded treatment groups (such as DXZ + Ce@mPDA-C/P and D/Ce@mPDA-C/P), with the D/Ce@mPDA-C/P group showing the lowest non-heme iron content (Fig. [102]3B). Specifically, DXZ effectively reduced the accumulation of free iron in the heart tissues, which could potentially alleviate oxidative damage during ischemia-reperfusion. In contrast, there were no significant changes in heme iron levels (Fig. [103]3C), suggesting that DXZ mainly targeted non-heme iron without interfering with heme iron metabolism. Fig. 3. [104]Fig. 3 [105]Open in a new tab Evaluation of Iron Distribution, Oxidative Stress, and Cellular Apoptosis in the MIRI Mouse Model. (A) Quantitative analysis of total iron content, (B) non-heme iron content and (C) heme iron content in left ventricular (LV) tissue at Day 4 after MIRI. (D) Representative fluorescent staining images. Top row: DHE staining (red) for oxidative stress assessment, with DAPI (blue) as a nuclear counterstain. Second row: TUNEL staining (green) for apoptotic cells, with DAPI (blue). Third row: CD80 (green) and CD206 (red) staining for macrophage polarization assessment, with DAPI (blue). Fourth row: CD206 (red) and DAPI (blue). Bottom row: CD80 (green) and DAPI (blue). Scale bar = 100 μm. (E) Quantitative analysis of DHE relative fluorescence intensity. (F) Quantitative analysis of TUNEL-positive cell percentage. (G) Quantitative analysis of the CD206/CD80-positive cell ratio. Data are presented as the mean ± standard deviation (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ns ≥ 0.05 Next, we assessed the oxidative stress levels, apoptosis, and inflammation in myocardial tissues using immunofluorescence (Fig. [106]3D). By employing dihydroethidium (DHE) staining, we detected oxidative stress markers in heart tissues, while TUNEL staining was used to observe apoptotic cells. Quantitative analysis showed that all treatment groups significantly reduced oxidative stress levels and apoptosis in the heart compared to the control group, with the most pronounced effects observed in the D/Ce@mPDA-C/P group, where both oxidative stress and apoptosis were most effectively suppressed (Fig. [107]3E, F). When comparing different treatment strategies, the CHP-modified Ce@mPDA-C group exhibited significantly better therapeutic effects than the unmodified Ce@mPDA group. As a cardiac-targeting peptide, CHP effectively enhanced the accumulation of CeO[2]@mPDA nanoparticles in the heart injury region, improving their targeting capability and making this strategy particularly significant in alleviating MIRI-induced myocardial damage. In comparison, the TPP-modified Ce@mPDA-C/P group demonstrated even better therapeutic efficacy. TPP facilitates the precise targeting of mitochondria, which are the primary sites of oxidative stress and ferroptosis. The TPP modification allowed CeO[2] nanoparticles not only to accumulate in the injured myocardium but also to enter the mitochondria effectively, enhancing their antioxidant effects and reducing mitochondrial oxidative damage and ferroptosis, ultimately contributing to better cardiac function recovery. Additionally, when DXZ was combined with Ce@mPDA-C/P nanoparticles, the therapeutic effects were significantly improved. In the D/Ce@mPDA-C/P group, the synergistic effect between DXZ and CeO[2] showed stronger efficacy compared to the DXZ + Ce@mPDA-C/P group. DXZ chelated free iron, lowering its levels in heart tissues, while CeO[2], with its potent antioxidant properties, inhibited oxidative stress. The combined effect of DXZ and CeO[2] significantly reduced myocardial apoptosis and oxidative damage, promoting heart repair. The D/Ce@mPDA-C/P group demonstrated superior therapeutic efficacy compared to the DXZ + Ce@mPDA-C/P group, highlighting that the mitochondrial-targeted delivery of DXZ loaded on CeO[2] nanoparticles enhances its therapeutic outcomes. Inflammation is a critical factor that exacerbates myocardial injury and plays a key role in myocardial repair [[108]29–[109]31]. Eliminating ROS in the myocardium helps modulate inflammation [[110]32]. To assess this, we analyzed the presence of CD206^+ and CD80^+ cells in myocardium using immunofluorescence staining (Fig. [111]3D), while CD206 and CD80 are markers often associated with M2 and M1 macrophages, respectively. The CD206/CD80 ratio (Fig. [112]3G) revealed that, in the saline group, CD80^+ macrophages dominated (with a lower CD206/CD80 ratio). In contrast, the Ce@mPDA-C, Ce@mPDA-C/P, and DXZ + Ce@mPDA-C/P groups showed a significant increase in the CD206/CD80 positive cells ratio. The D/Ce@mPDA-C/P group exhibited the highest CD206/CD80 positive cells ratio, indicating that these nanoparticles not only reduced oxidative stress and apoptosis but also promoted an anti-inflammatory microenvironment in the damaged myocardium. To further corroborate the anti-inflammatory effects of our nanoparticles and strengthen our conclusions, we performed quantitative real-time PCR (qRT-PCR) to assess the expression levels of the pro-inflammatory cytokines IL-1β and TNF-α (Figure S4). The results indicate that in the saline group, the expression levels of IL-1β and TNF-α were significantly elevated compared to the control group. Treatment with Ce@mPDA-C/P nanoparticles led to a marked reduction in the expression levels of both cytokines, and combination treatment with DXZ + Ce@mPDA-C/P further reduced cytokine expression. Notably, the D/Ce@mPDA-C/P group exhibited the most significant suppression of IL-1β and TNF-α, indicating that this formulation had the strongest anti-inflammatory effect. These findings confirm the ability of the nanoparticles to attenuate the inflammatory response in the damaged myocardium. Mechanistic validation of therapeutic effects To elucidate the mechanisms underlying the therapeutic effects of the hierarchical targeting system, we evaluated ferroptosis-related protein expression, mitochondrial ultrastructure, and transcriptomic changes. Western blot analysis revealed significant differences in ACSL4 and GPX4 expression across the treatment groups (Fig. [113]4A). In the Saline group, ACSL4, a ferroptosis marker, was significantly upregulated, while GPX4, a critical anti-ferroptotic protein, was markedly downregulated. Treatment with Ce@mPDA nanoparticles resulted in significant improvements, with ACSL4 levels decreasing and GPX4 levels increasing across all treatment groups. The Ce@mPDA-C group demonstrated enhanced efficacy compared to Ce@mPDA, with more pronounced suppression of ACSL4 and elevation of GPX4, attributed to CHP modification. The Ce@mPDA-C/P group exhibited further improvements due to TPP modification, which strengthened mitochondrial targeting, leading to lower ACSL4 and higher GPX4 expression. The DXZ + Ce@mPDA-C/P group ranked second in efficacy, significantly reducing ACSL4 expression and partially restoring GPX4 levels. The D/Ce@mPDA-C/P group achieved the best outcomes, with ACSL4 expression nearly suppressed and GPX4 restored to levels close to those of the control group (Fig. [114]4B, C). Fig. 4. [115]Fig. 4 [116]Open in a new tab Mechanistic analysis of the effects of different treatment groups on MIRI. (A) Western blot analysis of GPX4 and ACSL4 protein expression in infarcted myocardium on day 4 after treatment; (B) quantitative analysis of relative GPX4 protein and (C) ACSL4 protein expression (n = 8); (D) TEM images showing mitochondrial ultrastructure of myocardial cells in different groups; (E) GO (left, Biological Process [BP] and Cellular Component [CC]) and KEGG (right) pathway enrichment analysis comparing the Saline group with the Ce@mPDA-C/P group; (F) GO (left) and KEGG (right) pathway enrichment analysis comparing the Saline group with the D/Ce@mPDA group. Data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ns ≥ 0.05 Transmission electron microscopy revealed notable differences in mitochondrial ultrastructure among the groups (Fig. [117]4D). In the Saline group, mitochondria displayed severe fragmentation, disrupted cristae, and vacuolation, indicative of extensive oxidative stress and ferroptosis. Treatment with Ce@mPDA-based nanoparticles partially restored mitochondrial integrity, reducing fragmentation and improving cristae structure. The DXZ + Ce@mPDA-C/P group exhibited improved mitochondrial repair, with clearer cristae structure and significantly reduced vacuolation, reflecting enhanced protection against oxidative stress and ferroptosis. In contrast, the D/Ce@mPDA-C/P group showed the most substantial recovery, with mitochondrial morphology in most cells appearing nearly normal, though electron density had not fully returned to normal levels, indicating residual effects of the injury. RNA sequencing revealed significant differences in gene expression among the Saline, Ce@mPDA-C/P, and D/Ce@mPDA-C/P groups. Gene Ontology (GO) enrichment analysis indicated that Ce@mPDA-C/P significantly enhanced biological processes (BP) related to cardiac protection, including cardiac muscle contraction, regulation of neutrophil migration, mitochondrial membrane potential, inflammatory response, and oxygen level response. These findings suggest that Ce@mPDA-C/P alleviates myocardial ischemia-reperfusion injury (MIRI) by modulating inflammation, improving mitochondrial function, and stabilizing oxygen metabolism. Similarly, KEGG pathway enrichment analysis revealed key pathways such as oxidative phosphorylation, HIF-1 signaling, fatty acid metabolism, TNF signaling, and extracellular matrix (ECM)-receptor interaction. These pathways emphasize the antioxidative and anti-inflammatory properties of Ce@mPDA-C/P, collectively contributing to the preservation of cardiac function under MIRI stress (Fig. [118]4E). In comparison, D/Ce@mPDA-C/P exhibited a broader range of biological effects. GO enrichment analysis demonstrated significant regulation of macrophage chemotaxis, apoptotic mitochondrial changes, mitochondrial autophagy, reactive oxygen species metabolism, and ECM assembly. KEGG pathway enrichment analysis further highlighted the unique mechanisms of D/Ce@mPDA-C/P. Notably, unlike Ce@mPDA-C/P, D/Ce@mPDA-C/P showed significant enrichment in ferroptosis-related pathways in KEGG analysis (Fig. [119]4F). This indicates that even in ferroptosis-targeted nanotherapeutic systems, the additional delivery of DXZ still plays a significant role in mitigating myocardial ferroptosis. Long-term evaluation of myocardial remodeling and cardiac function at Day 28 Post-MIRI To assess the long-term therapeutic efficacy of different nanoparticle-based strategies, myocardial remodeling and cardiac function were evaluated on day 28 after MIRI. Key parameters included myocardial fibrosis, left ventricular remodeling, and functional recovery. Masson’s trichrome and Sirius Red staining (Fig. [120]5A) were performed to visualize and quantify myocardial fibrosis. The saline group exhibited significant myocardial fibrosis, with extensive collagen deposition, as indicated by the large areas of blue and red staining in Masson and Sirius Red staining, respectively. In contrast, treatment groups receiving Ce@mPDA-based nanoparticles showed a marked reduction in fibrosis. Among these, the D/Ce@mPDA-C/P group demonstrated the most substantial decrease in fibrotic regions, approaching levels observed in the sham group. Fig. 5. [121]Fig. 5 [122]Open in a new tab Long-Term Evaluation of Myocardial Remodeling and Cardiac Function at Day 28 Post-MIRI. (A) Representative images of Masson’s trichrome staining (top row), Sirius Red staining (middle row) of myocardial tissue sections and polarized light images (bottom row) of Sirius Red staining, scale bars 1 mm (Masson’s and Sirius Red staining) and 100 μm (polarized light). (B) Quantitative analysis of collagen volume fraction from Masson’s staining. (C) Quantitative analysis of collagen I/III ratio from polarized light images. (D) Representative echocardiographic M-mode images illustrating cardiac function in different treatment groups. E-J) Quantitative echocardiographic parameters: (E) LV internal diameter at systole (LVIDs), (F) end-systolic volume (ESV), (G) LV internal diameter at diastole (LVIDd), (H) end-diastolic volume (EDV), (I) fractional shortening (FS), and (J) ejection fraction (EF). Data are presented as the mean ± standard deviation (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ns ≥ 0.05 Quantitative analysis of collagen volume fraction (CVF, Fig. [123]5B) and the collagen type I/III ratio (Fig. [124]5C) confirmed these findings. The collagen type I/III ratio plays a crucial role in determining the structural and functional properties of the extracellular matrix (ECM) [[125]33]. Type I collagen provides tensile strength and rigidity to the myocardium, while type III collagen contributes to elasticity and compliance [[126]34, [127]35]. An elevated collagen I/III ratio, as observed in the saline group, indicates pathological remodeling of the ECM, leading to increased myocardial stiffness and impaired cardiac function. The reduction in the collagen I/III ratio in the Ce@mPDA-treated groups, particularly in the D/Ce@mPDA-C/P group, suggests that this treatment mitigates pathological ECM remodeling, thereby potentially improving myocardial elasticity and overall cardiac function. This highlights the therapeutic potential of Ce@mPDA in addressing fibrosis and restoring ECM balance following myocardial ischemia-reperfusion injury. Echocardiographic analysis was conducted to evaluate cardiac function recovery on day 28 (Fig. [128]5D). The saline group exhibited severe ventricular dilation and reduced cardiac contractility, as reflected by increased left ventricular internal diameters during systole (LVIDs, Fig. [129]5E) and diastole (LVIDd, Fig. [130]5G). In addition, end-systolic volume (ESV, Fig. [131]5F) and end-diastolic volume (EDV, Fig. [132]5H) were significantly elevated, indicating poor ventricular remodeling. Treatment with Ce@mPDA nanoparticles effectively mitigated these pathological changes. Specifically, the D/Ce@mPDA-C/P group demonstrated near-normal values for LVIDs, LVIDd, ESV, and EDV, indicating superior preservation of left ventricular structure compared to other treatment groups. Left ventricular fractional shortening (FS, Fig. [133]5I) and ejection fraction (EF, Fig. [134]5J) were analyzed to assess myocardial contractility. Both FS and EF were significantly reduced in the saline group compared to the sham group (p < 0.001). Among the treatment groups, the D/Ce@mPDA-C/P group exhibited the most substantial improvements in FS and EF, surpassing the therapeutic effects of DXZ + Ce@mPDA-C/P and other groups. These findings highlight the enhanced cardiac contractility and functional recovery achieved by the D/Ce@mPDA-C/P formulation. The superior efficacy of the D/Ce@mPDA-C/P group can be attributed to its hierarchical targeting strategy. The combination of CHP and mitochondrial-targeting TPP modifications allowed precise delivery of Ce@mPDA nanoparticles to the myocardium and mitochondria, where oxidative stress and ferroptosis are most prominent. Additionally, DXZ-loaded nanoparticles effectively chelated free iron, alleviating ferroptosis and oxidative damage, while CeO[2] nanoparticles scavenged ROS. This synergistic mechanism not only reduced fibrosis but also restored cardiac function to near-normal levels. Discussion This study presents an innovative strategy that addresses the dilemma of CeO[2] biological activity and delivery efficiency in injured myocardium. By using biomineralization, we combine small-sized CeO[2] (2–3 nm) with approximately 130 nm mPDA nanoparticles to form a composite system. The optimal size of nanoparticles for injured myocardium delivery is typically between 20 and 200 nm. Smaller nanoparticles are prone to accumulation or removal in organs such as the liver and kidneys, whereas larger nanoparticles are less likely to traverse the gaps between damaged endothelial cells [[135]36–[136]40]. Ce@mPDA composite material retains the small size properties of CeO[2] while achieving an appropriate particle size for efficient delivery to the injured myocardium [[137]9–[138]14]. With CHP and TPP modifications, the CeO[2] nanocatalyst-mesoporous polydopamine nanoparticle system is able to specifically target the mitochondria of the myocardium [[139]20, [140]21]. When loaded with DXZ for the treatment of MIRI, this system significantly enhances the therapeutic effect of DXZ. This finding has important clinical significance, as early intervention in the acute phase of MIRI is generally more effective than late intervention [[141]41, [142]42]. Clinically, sustained-release drugs are less commonly used to treat MIRI, as small molecule drugs can rapidly elevate drug concentrations in the bloodstream, leading to faster therapeutic effects. Sustained-release drugs are commonly used for medications that require maintaining a stable plasma concentration to prevent the recurrence of symptoms. Although nanoparticle-based drug delivery systems can improve drug delivery efficiency and bioavailability, if the delivery efficiency is insufficient or if the drug is not delivered to the key therapeutic site, it may fail to achieve the desired therapeutic effect [[143]43]. Our results further emphasize the crucial role of protecting myocardial mitochondria in the treatment of MIRI. Mitochondria are not only the energy centers of myocardial cells but also the main source of ROS generation [[144]44]. Under physiological conditions, an appropriate amount of ROS is essential for normal cell function. However, after MI, due to hypoxia, myocardial cells lose their ability to perform aerobic metabolism, leading to the accumulation of anaerobic metabolic products [[145]45]. During the subsequent reperfusion process, these accumulated metabolites react with oxygen to produce excessive ROS, resulting in oxidative damage. Moreover, during MI, the balance between oxidation and antioxidation is disrupted, particularly the expression and activity of GPX4, further exacerbating oxidative stress [[146]46, [147]47]. The downregulation of GPX4 in MIRI is primarily due to excessive oxidative stress. Excessive ROS depletes or inactivates GPX4, impairing its ability to maintain normal antioxidant function [[148]48]. Additionally, ROS accumulation activates redox signaling pathways such as the NF-κB (Nuclear Factor Kappa-B) pathway, which suppresses the expression of antioxidant genes, thereby reducing GPX4 synthesis [[149]49]. Studies have shown that during ischemia-reperfusion, cells may degrade GPX4 through autophagy, leading to further reduction in its levels [[150]50]. In addition to ROS accumulation and the imbalance of the antioxidant system, Fe^2+ plays a crucial role in MIRI [[151]51]. Ferroptosis is a form of cell death triggered by iron-ion-catalyzed lipid peroxidation. After MI, myocardial hemorrhage often occurs, and the degradation of hemoglobin releases iron ions, increasing the iron concentration in the myocardium [[152]24, [153]25]. Fe^2+ catalyzes the conversion of H[2]O[2] to •OH through the Fenton reaction, exacerbating lipid peroxidation and further inducing ferroptosis [[154]51]. Inhibiting ferroptosis depends on both iron chelation and the restoration of the oxidation-antioxidation balance [[155]46, [156]47]. The downregulation and depletion of antioxidant enzymes like GPX4 is one of the main reasons for weakened antioxidant capacity following MIRI [[157]49, [158]50]. Relying solely on endogenous antioxidant mechanisms is insufficient to cope with acute myocardial injury, thus external supplementation of antioxidant-functional CeO[2] nanozymes is an effective strategy to restore the oxidation-antioxidation balance. CeO[2] exhibits catalytic activity CAT and SOD, effectively scavenging H[2]O[2] and O[2]⁻ [[159]52, [160]53]. Additionally, iron chelators can effectively inhibit the production of •OH [[161]17, [162]18]. The combination of CeO[2] and iron chelators can further eliminate various types of ROS, inhibit ferroptosis, and protect myocardial cells. Our D/Ce@mPDA-C/P system targets the delivery of DXZ and CeO[2] nanocatalysts to the mitochondria of injured myocardium, reducing ROS production in the MIRI mouse model and upregulating GPX4 expression, thereby protecting myocardial cells and reducing apoptosis. Additionally, this system can modulate the inflammatory microenvironment of the injured myocardium, promoting myocardial repair. After 28 days of MIRI, our system effectively protected the cardiac function of the mice and reduced myocardial fibrosis. This approach providing a promising framework for the development of more effective therapies for MIRI. Future studies can build on these findings to further refine nanomedicine strategies for myocardial repair and recovery. Conclusion In this study, we successfully developed a hierarchical targeting delivery system based on CeO[2]-loaded mPDA nanoparticles for the treatment of MIRI. By incorporating CHP and mitochondrial-targeting TPP, the system was able to effectively target the damaged myocardium and mitochondria, significantly enhancing the therapeutic efficacy of the loaded drug, DXZ. The combination of CeO[2] nanozymes, mesoporous polydopamine nanoparticles, and targeted delivery strategies provides a promising approach for the treatment of myocardial ischemia-reperfusion injury. The synergistic effects of DXZ and CeO[2], coupled with efficient targeting to both the myocardium and mitochondria, significantly improve therapeutic outcomes, reduce side effects, and may offer a pathway to more effective clinical treatments for MIRI and other cardiovascular diseases related to oxidative stress and ferroptosis. Methods Materials Cerium nitrate hexahydrate (Ce(NO[3])[3]·6H[2]O), dopamine hydrochloride were purchased from Sigma-Aldrich. The NH[2]-PEG[2000]-CHP(CSTSMLKAC) and NH[2]-PEG[2000]-TPP was synthesized by Ruixi (Xi’an, China). MitoTracker Green, MitoSOX Red, and other cell culture reagents were obtained from Invitrogen (USA). Centrifuge tubes were purchased from Guangzhou Jet Bio-Filtration Co., Ltd. Cell culture dishes and inserts were obtained from NEST Biotechnology Co. Ltd. (Wuxi, China). All antibodies were purchased from Abcam (USA), and gel preparation kits were purchased from New Cell Molecular (China, Express Cast). Cell culture slides were obtained from Kirgen Bioscience (Shanghai) Co., Ltd. The cell culture medium was supplemented with 10% fetal bovine serum (BDBIO, HangZhou, China) and 1% penicillin/streptomycin (Shanghai Chuanqiu Biotechnology Co., Ltd., China). Cellular proteins were extracted using an ultrasonic cell crusher (Open, 3s; Close, 5s; JY92-IIN, Scientz, Shanghai, China). Synthesis of CeO[2] -Loaded mPDA (Ce@mPDA) Nanoparticles: The synthesis of mPDA was based on a previously reported method. Dissolve 0.15 g of dopamine and 0.1 g of F127 in 10 mL of 50% ethanol solution and mix thoroughly under stirring. Add 250 µL of tetramethylbenzidine to the mixture and allow the reaction to proceed at room temperature for 30 min. Next, add 375 µL of 2 M ammonium hydroxide and continue stirring at room temperature for another 2 h. After the reaction, wash the product twice with 50% ethanol and redisperse it in 2 mL of ethanol for future use. To synthesize Ce@mPDA, add the mPDA product to 30 mL of water and stir for 10 min. Dissolve 6.3 mg of cerium nitrate hexahydrate in 1 mL of ethylene glycol and add this solution to the dispersion. Stir for another 10 min, then add 250 µL of ammonium hydroxide. Allow the reaction to proceed at 60 °C for 3 h. Wash the resulting product twice with ethanol, and the Ce@mPDA nanoparticles are ready for further use. For experimental use, all Ce@mPDA nanoparticles were PEG-modified. Weigh 5 mg of NH[2]-PEG[2000] and add it to the dispersion of Ce@mPDA nanoparticles (1 mg). Adjust the pH of the reaction mixture to 10 using ammonium hydroxide. Stir the reaction mixture continuously for 24 h to ensure efficient PEG binding to the nanoparticle surface. After the reaction, purify the product three times using a 30 kD ultrafiltration membrane to remove unbound PEG molecules and by-products. The purified PEG-modified Ce@mPDA nanoparticles were collected and stored for subsequent experiments. Surface modification of nanoparticles To enhance targeting capabilities, Ce@mPDA nanoparticles were modified with NH[2]-PEG[2000]-CHP. Weigh 5 mg of NH[2]-PEG[2000]-CHP and add it to the dispersion of Ce@mPDA nanoparticles (1 mg). Adjust the pH of the reaction mixture to 10 using ammonium hydroxide. Allow the reaction to proceed under continuous stirring for 24 h. Afterward, purify the product three times using a 30 kD ultrafiltration membrane. Collect the purified product, designated as Ce@mPDA-C, for further use. For mitochondrial targeting, modify Ce@mPDA nanoparticles with NH[2]-PEG[2000]-TPP and NH[2]-PEG[2000]-CHP. Add 5 mg each of NH[2]-PEG[2000]-TPP and NH[2]-PEG[2000]-CHP to the Ce@mPDA dispersion. Adjust the pH to 10 with ammonium hydroxide and stir continuously for 24 h. After the reaction, purify the product three times using a 30 kD ultrafiltration membrane. Collect the purified product, designated as Ce@mPDA-C/P, and dialyze it. Store the final nanoparticles at 4 °C until further use. Characterization of nanoparticles The morphology and size of the nanoparticles were characterized by transmission electron microscopy (TEM, JEOL JEM-1400, Japan). The particle size distribution was analyzed using dynamic light scattering (DLS, Malvern Zetasizer, UK), and the surface charge was measured by ζ-potential analysis. The elemental composition and oxidation states of CeO[2] were determined by X-ray photoelectron spectroscopy (XPS, Thermo Fisher ESCALAB 250Xi, USA). Loading and release of DXZ onto nanoparticles DXZ was loaded onto the nanoparticles via a simple adsorption method. Specifically, 10 mg of nanoparticles were mixed with an aqueous solution of DXZ (1 mg/mL) and incubated at room temperature for 24 h to ensure sufficient adsorption. The DXZ-loaded nanoparticles were then purified by centrifugation three times to remove any unbound DXZ. The concentration of DXZ in the supernatant was determined by high-performance liquid chromatography (HPLC), and the drug loading efficiency was calculated accordingly. To evaluate the release profile of DXZ from Ce@mPDA-C/P nanoparticles, a dialysis method was employed. Specifically, 5 mg of DXZ-loaded Ce@mPDA-C/P nanoparticles were dispersed in 2 mL of phosphate-buffered saline (PBS, pH 7.4) and placed into a dialysis bag with a molecular weight cutoff of 10 kD. The dialysis bag was immersed in 50 mL of PBS (pH 7.4) containing 0.5% Tween 80 to maintain sink conditions, and the system was kept at 37 °C under constant stirring at 100 rpm to simulate physiological conditions. At predefined time intervals (e. g., 0, 1, 2, 4, 8, 12 h), 1 mL of the external release medium was sampled, and an equal volume of fresh PBS was added to maintain the total volume. The collected samples were analyzed by HPLC to determine the concentration of DXZ released at each time point. The cumulative drug release percentage was calculated based on the initial drug loading. All experiments were conducted in triplicate, and the average release profile was reported. ROS scavenging assay The H[2]O[2] scavenging ability is assessed using the TMB colorimetric method, where nanoparticles are added to PBS containing different concentrations of H[2]O[2]. After incubation at room temperature, TMB is added to initiate the color reaction, and the absorbance at 652 nm is measured to calculate the scavenging rate. The •OH scavenging ability is evaluated through the Fenton reaction system, where PBA is used as a probe to capture hydroxyl radicals, forming a fluorescent product. The fluorescence intensity is measured at an excitation wavelength of 320 nm and an emission wavelength of 420 nm to calculate the scavenging rate. The ·O[2]⁻ scavenging ability is determined using the xanthine-xanthine oxidase (XOD) system, with NBT as a probe. The absorbance of the resulting formazan is measured at 560 nm to calculate the scavenging rate. Analysis of nanoparticle myocardial delivery The experimental protocol involves labeling nanoparticles with NH[2]-PEG[2000−]ICG and conducting small animal in vivo imaging after MI reperfusion. NH[2]-PEG[2000]-ICG is added during the nanoparticle modification process to obtain ICG-labeled nanoparticles, which are then purified to remove unbound components. After establishing an MI model in mice and restoring reperfusion, the labeled nanoparticles are administered via tail vein injection. At 4 h post-injection, the mice are euthanized, and heart tissues are collected for fluorescence imaging using a small animal imaging system to evaluate nanoparticle distribution and targeting to the injured myocardium. In Vitro Cellular studies In all cell experiments, the dosage of nanoparticles was maintained at 50 µg/mL (calculated based on the mass of mPDA). H9C2 cardiomyocytes were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO[2] incubator. To evaluate the cellular uptake and mitochondrial targeting, H9C2 cells were incubated with Cy7-labeled Ce@mPDA, Ce@mPDA-C, or Ce@mPDA-C/P nanoparticles for 4 h, followed by washing and staining with MitoTracker Green. Fluorescent images were captured using a confocal microscope (Leica TCS SP8, Germany). The labeling method of nanoparticles with Cy-7 is consistent with the aforementioned ICG labeling method. To assess the ROS-scavenging effect, H9C2 cells were treated with the nanoparticles, and mitochondrial ROS levels were measured by flow cytometry using MitoSOX Red staining. The anti-apoptotic effect was evaluated by Annexin V/PI staining, and apoptotic cells were quantified using flow cytometry. To investigate ferroptosis inhibition, the expression of GPX4 and ACSL4 was analyzed by Western blotting. Briefly, cells were lysed in RIPA buffer, and protein levels were quantified using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes, which were probed with anti-GPX4 and anti-ACSL4 antibodies. In vivo studies All animal procedures were approved by the Ethics Committee of Tongji University Affiliated Eastern Hospital. MIRI was induced in C57BL/6 mice (20 g, female) by ligating the left anterior descending coronary artery for 30 min, followed by reperfusion. The nanoparticles were intravenously injected into the mice at a dose of 10 mg/kg (calculated based on the mass of mPDA). The amount of DXZ used in DXZ + Ce@mPDA-C/P group is consistent with the DXZ content used in D/Ce@mPDA-C/P group. At 4 days post-injection, heart tissues were collected and analyzed by immunofluorescence, and TUNEL assay to evaluate oxidative stress, apoptosis, and inflammation. Iron Content Analysis in Left Ventricular Myocardium The left ventricle (LV) was defined as the thicker-walled region in the lower-left portion of the heart, with tissue samples carefully collected to include areas of MIRI, adjacent healthy myocardium, and regions near the apex. The interventricular septum and left atrium were excluded to ensure consistency. Total iron concentration was measured by inductively coupled plasma-optical emission spectroscopy. LV myocardial non-haem iron concentration was measured by a modified ferrozine-based colorimetric method. A homogenized tissue sample was mixed with an equal volume of extraction buffer (25% trichloroacetic acid, 4% sodium pyrophosphate) and heated at 65 °C for 2 h. After centrifugation, 50 µL of the supernatant was combined with 150 µL of ferrozine solution (50 mmol/L ascorbic acid, 1.7 mmol/L ferrozine, 1.8 mol/L sodium acetate), and the absorbance was measured at 562 nm using a microplate reader. For LV myocardial haem concentration, the homogenized sample was treated with 2 mol/L oxalic acid and heated at 95 °C for 30 min to release fluorescent porphyrin, which was measured at 360 nm excitation and 590 nm emission on the same microplate reader. Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA from the MIRI region of each group was extracted using the TRIzol reagent. RNA was then reverse-transcribed into complementary DNA using a Reverse Transcription Kit. Quantitative real-time PCR was conducted with the SYBR Premix Ex Taq II Kit. The primers were designed based on cDNA sequences obtained from NCBI, and GAPDH was used as an internal reference gene. The qRT-PCR reaction mixture consisted of 2 µL cDNA, 2 µL primers (1 µmol/L), 10 µL SYBR green dye (2×), and 6 µL nuclease-free water, making a total volume of 20 µL. The reaction conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 42 cycles of 95 °C for 3 s, 55 °C for 30 s, and 72 °C for 30 s. This was followed by 95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s. The PCR was confirmed by the product melting curve, and the Ct values of the target gene and GAPDH of the sample were obtained based on the PCR curve. Sequence of Primers for q-PCR was provided at Supported Information. The relative expression level for target genes were performed with a value of 2^-ΔΔCt. Long-term evaluation of cardiac function Cardiac function was evaluated at 28 days post-MIRI using echocardiography (Philips EPIQ5 system). Left ventricular internal diameters (LVIDs, LVIDd), end-systolic volume (ESV), and end-diastolic volume (EDV) were measured. Left ventricular fractional shortening (FS) and ejection fraction (EF) were calculated to assess myocardial contractility. EDV = 7*LVIDd^3/(2.4 + LVIDd); ESV = 7*LVIDs^3/(2.4 + LVIDs); FS = (LVIDd-LVIDs)/LVIDd; EF = (EDV-ESV)/EDV. After the echocardiographic assessment of cardiac function, the mice were euthanized, and heart tissues were collected for histological analysis. Masson’s trichrome staining and Sirius Red staining were performed on the heart sections to evaluate fibrosis. Polarized light microscopy was used to visualize the collagen I/III ratio, and ImageJ software was utilized for the quantification of collagen deposition and the collagen I/III ratio. Statistical analysis Data are presented as the mean ± standard deviation. Statistical comparisons were performed using one-way analysis of variance followed by Tukey’s post-hoc test. A p-value of < 0.05 was considered statistically significant. Electronic supplementary material Below is the link to the electronic supplementary material. [163]Supplementary Material 1^ (508.3KB, docx) Abbreviations MIRI Myocardial Ischemia-Reperfusion Injury MI Myocardial Infarction PCI Percutaneous Coronary Intervention CeO[2] Cerium Oxide PDA Polydopamine mPDA Mesoporous Polydopamine DXZ Dexrazoxane CHP Cardiac Homing Peptide TPP Triphenylphosphine ROS Reactive Oxygen Species GPX4 Glutathione Peroxidase 4 H/R Hypoxia/Reoxygenation DLS Dynamic Light Scattering TEM Transmission Electron Microscopy XPS X-ray Photoelectron Spectroscopy CAT Catalase SOD Superoxide Dismutase DHE Dihydroethidium CVF Collagen Volume Fraction ECM Extracellular Matrix LVIDs Left Ventricular Internal Diameter during Systole LVIDd Left Ventricular Internal Diameter during Diastole FS Fractional Shortening EF Ejection Fraction EDV End-Diastolic Volume ESV End-Systolic Volume NF-κB Nuclear Factor Kappa-B Author contributions K.Z., K.W., R.Z. contributed equally to this work. K.Z., K.W., Y.S. conceived and designed the project. K.Z., K.W., R.Z., W.W., B.Y. carried out the cell experiments. K.Z., K.W., R.Z. carried out the animal experiments. K.Z., K.W. analyzed the data. K.Z. drafted the manuscript. Y.S. and J.Z. supervised the research and revised the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Training plan for discipline leaders of Shanghai Pudong New Area Health Commission (PWRd2020-09), Top-level Clinical Discipline Project of Shanghai Pudong District (grant/award number: PWYgf 2021-01). Data availability No datasets were generated or analysed during the current study. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ke Zhu, Kun Wang and Rongting Zhang contributed equally to this work. Contributor Information Jun Zhao, Email: petcenter@126.com. Yunli Shen, Email: shenyunli2011@163.com. References