Hydrogen Peroxide‐Responsive Nanoparticle Reduces Myocardial Ischemia/Reperfusion Injury

Figures

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  Figure 1. Chemical and biological properties of PVAX and HPOX nanoparticles. A, Chemical structure of antioxidant copolyoxalates, PVAX and HPOX. B, Scavenging of H2O2 by PVAX and HPOX nanoparticles. C, Sensitivity of PVAX and HPOX nanoparticles to H2O2 based on peroxalate chemiluminescence. D, Inhibitory effects of PVAX and HPOX nanoparticles on ROS generation in PMA‐stimulated cells. E, Inhibitory effects of PVAX and HPOX nanoparticles on H2O2‐induced apoptotic cell death. PMA indicates phorbol‐12‐myristate‐13‐acetate; ROS, reactive oxygen species.

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  Figure 2. The therapeutic effects of PVAX and HPOX nanoparticles on hind limb I/R injury. A and B, Quantification of caspase‐3 (A) and PARP activity (B) *P<0.05 vs sham of each group; † P<0.05 vs HPOX I/R. (n=4/group). Mann‐Whitney test was used. C, Expression of inflammatory markers TNF‐α and MCP‐1 in hind limb I/R injury. D and E, Quantification of TNF‐α (D) and MCP‐1 (E) mRNA level, *P<0.05 vs sham of each group; † P<0.05 vs HPOX I/R. (n=4/sham group, n=6/I/Rgroup). ANOVA and Bonferroni test were used. F, Representative images of gastrocnemius muscle tissues stained by hematoxylin and eosin (×200). I/R indicates ischemia/reperfusion; MCP‐1, monocyte chemotactic protein‐1; TNF‐α, tumor necrosis factor‐α.

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  Figure 3. The therapeutic effect of PVAX on H2O2‐stimulated adult rat cardiomyocytes in vitro. A, Protective effects of PVAX nanoparticles on H2O2‐stimulated cardiomyocytes. (n=3/group). Mann‐Whitney test was used. B, Expression of TNF‐α and MCP‐1. The images are representatives of 4 experiments. C through F, Quantification of TNF‐α (C), MCP‐1 (D), NOX2 (E), and NOX4 (F). *P<0.05 vs sham of each group. (n=4/group). The Mann‐Whitney test was used. MCP‐1 indicates monocyte chemotactic protein‐1; NOX2, NADPH oxidase 2; NOX4, NADPH oxidase 4; TNF‐α, tumor necrosis factor‐α.

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  Figure 4. Effect of different PVAX nanoparticle administration methods on I/R‐induced cardiac dysfunction. A and B, Cardiac functions after I/R were evaluated after vehicle (Veh), daily injection of PVAX (3 mg/kg per day for 2 weeks) (2W), or single injection of PVAX (3 mg/kg) (1). Stroke work (SW) (A) was determined using PV loop analysis. Fractional shortening (FS) (B) was measured using echocardiography. *P<0.05 vs Veh. NS=not significant (n=6/veh, n=4/PVAX 2W and 1 group). The Kruskal‐Wallis and Dunn tests were used. I/R indicates ischemia/reperfusion.

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  Figure 5. Effect of PVAX and PLGA nanoparticles on I/R‐induced cardiac dysfunction. A, Cardiac output (CO), (B) stroke work (SW), and (C) fractional shortening (FS) 2 weeks after I/R in mice after PLGA or PVAX (3 mg/kg, single injection). *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R. (n=5/sham group, n=6/IR group). ANOVA and a Bonferroni test were used. D, Representative images of heart tissues after I/R injury after TTC staining (top) or fluorescent microsphere administration (bottom). E, Representative heart tissue sections stained with TTC solution. Red‐colored regions in the TTC‐stained sections indicate nonischemic areas; pale‐colored regions indicate ischemic portions of the heart. F, Quantification of infarction area (IA)/area at risk (AAR) *P<0.05 (n=4/PLGA, n=5/PVAX). Student t test was used. I/R indicates ischemia/reperfusion; TTC, triphenyltetrazolium chloride.

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  Figure 6. Anti‐inflammatory and antiapoptotic activities of PVAX nanoparticles after cardiac I/R injury. A, Quantification of caspase‐3 activity assay. *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R (n=4/sham group, n=5 per I/R group). ANOVA and a Bonferroni test were used. B, Representative images of TUNEL staining. TUNEL, green; nuclei, blue; α‐actinin, red. C, Quantification of TUNEL‐positive cells/total cells. *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R. (n=4/sham group, n=5 per I/R group). ANOVA and a Bonferroni test were used. D, Expression of inflammatory markers TNF‐α and MCP‐1. E and F, Quantification of TNF‐α (E) and MCP‐1 (F) mRNA level. *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R. (n=4/sham group, n=5 per I/R group). ANOVA and a Bonferroni test were used. I/R indicates ischemia/reperfusion; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling; TNF‐α, tumor necrosis factor‐α.

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  Figure 7. Antioxidant activities of PVAX nanoparticles after cardiac I/R injury. A, Representative image of DHE staining. DHE=red, nuclei=blue. B, Quantification of DHE‐positive nuclei. *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R (n=4/sham group, n=5 per I/R group). ANOVA and a Bonferroni test were used. C, Representative images of mRNA expression of NOX2 and NOX4. D and E, Quantification of NOX2 (D) and NOX4 (E) expression. *P<0.05 vs sham of each group; † P<0.05 vs PLGA I/R (n=4/sham group, n=5 per I/R group). ANOVA and a Bonferroni test were used. DHE indicates dihydroethidium; I/R, ischemia/reperfusion; NOX2, NADPH oxidase 2.