Single-atom Pt-doped ceria nanozymes mitigate myocardial ischemia reperfusion injury via cardiomyocyte-targeted uptake and suppression of reactive oxygen species

Abstract

The primary treatment for myocardial infarction (MI) is restoring blood flow to the obstructed coronary artery. However, this approach can paradoxically generate reactive oxygen species (ROS), leading to secondary ischemia-reperfusion (IR) injury. Multifunctional nanomaterials present a promising alternative for managing IR injury, offering benefits including cost-effectiveness, robust catalytic stability, and customizable properties that surpass traditional antioxidants. This study explores single-atom Pt-doped ceria nanozymes (Pt@CeNZ) with multi-enzyme mimetic functions facilitated by atomically dispersed Pt. The nanozymes effectively eliminate excess ROS in cardiomyocytes, thereby enhancing cell viability. Notably, Pt@CeNZ demonstrates significantly higher uptake in cardiomyocytes, underscoring its potential as a targeted nanotherapeutic for cardiac tissues. In vivo studies further confirm that Pt@CeNZ treatment substantially reduces infarct size and improves cardiac function following IR injury, without inducing long-term toxicity or inflammation. These findings position Pt@CeNZ as a highly promising heart-targeting nanotherapeutic with potential applications in the acute and long-term treatment of cardiac injuries.

Keywords: Antioxidation; Cardiomyocyte-targeted; Ceria nanozymes; Myocardial ischemia-reperfusion (IR) injury; Reactive oxygen species (ROS).

Graphical abstract

Fig. 1

Synthesis of single atom Pt-doped CeNZ, Pt@CeNZ. (A) Graphical representation, showing the synthesis of Pt@CeNZ. (B) Schematic illustrating the structure of Pt@CeNZ, indicating the presence of atomically dispersed Pt within the CeNZ lattice. (C) Representative FE-SEM images of Pt@CeNZ demonstrating its cube-like morphology; the inset shows the morphology of pure CeNZ. (D) HAADF-STEM image, revealing the internal structure of the Pt@CeNZ. (E) EDS mapping of Pt@CeNZ, displaying the uniform distribution of Pt, Ce, and O. (F) Location of single atom Pt sites on the surface of Pt@CeNZ with positions 1 and 2 highlighted. (G) Line profiles corresponding to positions from the TEM images. (H) Characteristic XRD spectra of Pt@CeNZ, showing no distinct Pt peaks, indicating that the Pt is atomically dispersed within the CeNZ lattice.

Fig. 2

Physicochemical properties and multi-enzymatic activities of Pt@CeNZ. (A) Raman spectra of Pt@CeNZ. (B) Pt L3-edge XANES spectra of Pt@CeNZ and its magnified view. (C) Pt L3-edge EXAFS spectra of Pt@CeNZ compared with Pt foil reference. (D) Wavelet transform (WT) images for the EXAFS spectra of Pt@CeNZ and Pt foil reference. (E) XPS analysis of Pt@CeNZ showing characteristic Ce3d peaks. (F) XPS analysis of Pt@CeNZ showing characteristic O1S peaks. (G) XPS analysis of Pt@CeNZ showing characteristic Pt4f peaks. (H) POD-mimicking activity of Pt@CeNZ. (I) CAT-mimicking activity of Pt@CeNZ. (J) SOD-mimicking activity of Pt@CeNZ. Data reported as mean ± SD (n = 3).

Fig. 3

Pt@CeNZ protected cardiomyocytes against oxidative injury. (A) Scheme of evaluating the cytocompatibility and cellular protective effects of Pt@CeNZ on cardiomyocytes. (B) Representative images of live/dead assay revealing cytocompatibility of Pt@CeNZ in a long-term treatment on NRCMs. (C) Quantification of percent live/dead cells. (D) Damage of NRCMs evaluated by the extracellular release of LDH. (E-G) Cellular protective effects of Pt@CeNZ from 30 min, 1, and 2 h co-treatment with H₂O₂ on H9c2 cells and cell viability determined by CCK-8 assay. ∗p < 0.05 versus H₂O₂ controls. (H) Cellular protective effects of Pt@CeNZ from 1 pretreatment before 30 min H₂O₂ treatment on H9c2 cells and cell viability was determined by CCK-8 assay. ∗p < 0.05 versus H₂O₂ controls. (I-J) Confirmation of the cytoprotective effects from Pt@CeNZ on NRCMs from co-treatment and pretreatment conditions. Cell viability as determined by CCK-8 assay. ∗p < 0.05 versus H₂O₂ controls. n = 3. (K) Anti-ferroptosis effects of Pt@CeNZ demonstrated using RSL3-induced ferroptosis. ∗p < 0.05 versus RSL3 controls. n ≥ 3.

Fig. 4

Pt@CeNZ suppressed intracellular ROS production in cardiomyocytes under oxidative stress. (A-B) 3 min co-treatment of Pt@CeNZ inhibited H₂O₂-induced ROS production in H9c2 cells as indicated by DCF fluorescent staining and intensity quantification. ∗p < 0.05 versus H₂O₂ controls. n = 3. (C-D) Flow cytometry results showing the ROS scavenging effects from 24 h pretreatment of Pt@CeNZ on H9c2 cells before 30 min incubation with H₂O₂ followed by DCF intensity analysis normalized to DMSO controls. ∗p < 0.05 versus H₂O₂ controls. n = 3. (E-F) Flow cytometry results showing the ROS scavenging effects from a 24 h pretreatment of Pt@CeNZ on H9c2 cells before exposure to normoxia and H/R conditions and quantification of the overlay intensity from different groups. (G-H) Intracellular ROS levels in NRCMs as evidenced by DCFDA assay following 24 h pretreatment and 1 h co-treatment of Pt@CeNZ. ∗p < 0.05 versus H₂O₂ controls. n = 4. (I) 24 h pretreatment of Pt@CeNZ inhibited hypoxia/reperfusion (H/R)-induced ROS production in NRCMs. ∗p < 0.05 versus H₂O₂ controls. n = 5. (J) 3 h pretreatment of Pt@CeNZ but not CeNZ inhibited H₂O₂-induced ROS production in NRCMs. ∗p < 0.05 versus H₂O₂ controls. n = 6.

Fig. 5

Preferential internalization and retention of Pt@CeNZ in cardiomyocytes (A) Fluorescent images of Dil dye-labeled Pt@CeNZ (Dil, red fluorescence). Scale bar = 500 μm. (B) Pt@CeNZ internalized into H9c2 after 24 h treatment following Pt@CeNZ removal. (C) Imaging flow cytometer images of H9c2 after 2 h treatment with 34.4 μg/mL Pt@CeNZ. (D) Quantification of Pt@CeNZ uptake efficiency in H9c2 by flow cytometry. ∗p < 0.05 versus H₂O₂ controls. n = 3. (E-F) Relative Dil-intensity quantification to confirm Pt@CeNZ uptake efficiency in H9c2 after 6 and 12 h incubation. ∗p < 0.05 versus H₂O₂ controls. n = 4. (G) High content screening images revealing long-term retention of Pt@CeNZ in NRCMs following 2 h treatment. (H-I) Relative Dil-intensity quantification to confirm longer retention of Pt@CeNZ in NRCMs following 6 and 24 h treatment. (J) Representative confocal images of NRCMs following 24 h Dil-Pt@CeNZ treatment.

Fig. 6

Pt@CeNZ protected cardiac function from ischemic-reperfusion injury (A) Representative images of Evans Blue & TTC staining at 24 h after the induction of MI. (B) Evans Blue & TTC staining quantification summary. ∗∗p < 0.05. n = 5. (C) Representative images M-mode of three groups at Base (4 h), 4 weeks post I/R. (D) Left ventricular ejection fraction (EF). Left fractional shortening (FS). Left ventricular internal dimension at end-diastole (LVIDd). Left ventricular internal dimension at end-systole (LVIDs). Septal wall thickness (SWT). Relative wall thickness (RWT). ∗p < 0.05 versus control. †p < 0.05 versus CeNZ. n = 7. (E) Representative images of the hemodynamic pressure and volume (PV) curve on steady state at 4 weeks post I/R injury. (F) Cardiac output. Stroke volume. Volume max (V max) at end-diastole. Maximal rate of pressure changes during systole (dP/dtmax). Minimal rate of pressure changes during diastole (dP/dtmin). ∗p < 0.05 versus control. †p < 0.05 versus CeNZ. n = 5.

Fig. 7

Pt@CeNZ alleviated cardiac cell death after myocardial infarction. (A) Representative images of cardiomyocytes stained with cTnT (green) and nanoparticles (red) at 7 days post-MI and their quantification summary. †p < 0.05 versus CeNZ. Scale bar: 200 μm. n = 5. (B) Representative images of MT staining at 4 weeks and quantification summary of a percentage of fibrosis and viable myocardium. ∗p < 0.05 versus PBS controls. †p < 0.05 versus CeNZ. Scale bar: 2000 μm. n = 5. (C-D) Representative images of cardiomyocytes stained with cTnT (green) and capillaries stained with CD31 (red) on the infarct zone, border zone at 4 weeks, and quantification summary. ∗p < 0.05 versus PBS controls. †p < 0.05 versus CeNZ. Scale bar: 200 μm. n = 5.

Fig. 8

In vivo biosafety of Pt@CeNZ. (A) Representative histology images of internal organs at 28 days after Pt@CeNZ administration in normal rats. Scale bars: 2000 μm. (B) Quantification summary of blood chemistry test and CBC results at day 0, 1, 3, 7, 21 and 28 after drug injection without myocardial infarction. n = 5.