Injured Myocardium‐Targeted Theranostic Nanoplatform for Multi‐Dimensional Immune‐Inflammation Regulation in Acute Myocardial Infarction

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  Scheme 1. A) Schematic overview of the approach to the preparation of multifunctional biomimetic HM4oRL nanoparticles (NPs). B) Schematic overview of the targeted effects of HM4oRL NPs on the damaged myocardium and as tools T1‐weighted MR imaging with a 9.4 T Micro MR scanner in a murine model of AMI. C) The proposed cardioprotective effects of HM4oRL NPs entail the suppression of ischemia‐induced cardiomyocyte pyroptosis, the preservation of microenvironmental cardiac metabolic homeostasis, and the modulation of cardiac monocyte/macrophage‐mediated inflammatory responses. CRMs: cardiac‐resident macrophages; TAC: tricarboxylic acid cycle.

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  Figure 1. Preparation and characterization of HM4oRL nanoparticles. A) Particle sizes, B) surface Zeta potential, and C) polydispersity index (PDI) of 4oRL, M4oRL, and HM4oRL nanoparticles. n = 3. D) TEM images of 4oRL, M4oRL, and HM4oRL nanoparticles in water, with HM4oRL nanoparticles negatively stained using phosphotungstic acid. E) FTIR spectra of 4‐OI, TA, 4oRL, and M4oRL nanoparticles. F) XPS survey scan spectra for 4oRL and M4oRL nanoparticles. (G) GSEA analysis of “focal adhesion” related enrichment plots derived from transcriptome sequencing in succinate activated neutrophils in vitro. n = 3. H) Top 20 DEGs in the “focal adhesion” gene set. I) Western blots of ITGB1, ITGA5, and ITGAX in neutrophil membrane (NM), activated neutrophil membrane (ANM), macrophage membrane (MM), hybrid membrane (HM), M4oRL, and HM4oRL nanoparticles. ITGB1: Integrin β‐1; ITGA5: Integrin α 5; ITGAX: Integrin α X. J) Antioxidant capacity of 4‐OI, 4oRL, M4oRL, MRL, and HM4oRL nanoparticles measured by ABTS free radical scavenging assays. n = 5. K) •OH and L) •O2 − scavenging capacity of TA, 4oRL, M4oRL, and MRL nanoparticles by the electron paramagnetic resonance spectra analysis. M) In vitro 4‐OI release…

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  Figure 2. HM4oRL NPs attenuate hypoxia‐induced cardiomyocyte pyroptosis and enhance resistance to the inflammatory microenvironment in vitro. A) CCK‐8 for the relative viability of H9c2 cells. n = 5. B) LDH activity derived from the media of hypoxia‐induced H9c2 cells. n = 4. C) Total SOD activity in H9c2 cells. n = 4. Mitochondrial superoxide was revealed by the red fluorescence of MitoSOX via E) confocal fluorescence images and D) corresponding analysis using ImageJ software. n = 4. Cell apoptosis was assessed by flow cytometry F), and the apoptosis rate was presented in bar graphs G). n = 4. H) Analytic result of intracellular ATP levels in H9c2 cells. n = 4. I) Western blotting analysis of NLRP3, Caspase‐1, GSDMD, and IL‐1β protein expression in hypoxia‐induced H9c2 cells after different treatments. J) Quantitative analysis of NLRP3, Cleaved‐Caspase‐1, Cleaved‐GSDMD, and Cleaved‐ IL‐1β protein levels in each group of H9c2 cells. n = 3. K) Schematic illustration of the H9c2 cell model subjected to continuous stimuli in a post‐hypoxic inflammatory medium. The conditioned medium (cm) was obtained from the supernatants collected after RAW 264.7 cells were stimulated with 200 ng/mL of LPS…

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  Figure 3. Targeted delivery and cardiac T1‐weighted MR imaging of HM4oRL in AMI model mice. A) In vivo NIRF fluorescence imaging of AMI or Sham‐operated mice at the indicated time point after intravenous administration of HMIrRL or MIrRL nanoparticles. B) Ex vivo NIRF fluorescence imaging of heart samples harvested from AMI or Sham‐operated mice at the indicated time point after the onset of coronary ligation, and C) quantification analysis of the fluorescence intensity in the infarcted area by ImageJ software. n = 3. * indicates p < 0.05. D) Heart samples were collected at the indicated time point after intravenous administration of HMIrRL in AMI mice. Subsequently, ex vivo NIRF fluorescence imaging of the collected heart and its slices was performed, followed by TTC staining of the corresponding slices. E) Representative fluorescence images and corresponding HE‐stained images of the infarct area, border zone, and remote area of the AMI heart after injection of HMIrRL nanoparticles. F) Heart samples were collected 12 h after intravenous administration of HMIrRL in AMI mice. Immunofluorescent staining was performed for cardiomyocytes (α‐actinin) and monocytes/macrophages (CD68, F4/80).…

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  Figure 4. HM4oRL suppresses the priming and activation of post‐MI NLRP3 inflammasome‐mediated pyroptosis. A) Schematic illustration of the experimental timelines for short‐term studies of MI mice. B) KEGG enrichment analyses of differentially expressed genes, obtained from transcriptome sequencing of infarcted myocardium in the Sham versus MI versus HM4oRL groups. n = 4. C) GSEA analysis of “Apoptosis”, “NF‐κB signaling pathway”, “NOD‐like receptor signaling pathway” related enrichment plots derived from transcriptome sequencing in MI versus Sham and HM4oRL versus MI groups. n = 4. D) Representative immunofluorescence micrographs of NLRP3+ and α‐actinin+ in the infarct regions, and E) quantification of NLRP3‐positive within α‐actinin‐positive cells of each group. n = 4. Green, α‐actinin; red, NLRP3; blue, DAPI. F) Western blotting of NLRP3, Caspase‐1, GSDMD, and IL‐1β protein expression in the infarcted myocardium from each group. G) Corresponding quantification of NLRP3, Cleaved‐Caspase‐1, Cleaved‐GSDMD, and Cleaved‐IL‐1β protein levels in each group. n = 3. H) Immunohistochemical staining and I) quantification of GSDMD‐positive fluorescence intensity in the infarcted area following…

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  Figure 5. HM4oRL preserves cardiac metabolic microenvironment homeostasis and inhibits pro‐inflammatory macrophage responses after MI. A) The numbers of identified metabolites from metabolomics of infarcted myocardium in each group 3 days after MI. B) The PLS‐DA score plot of the Sham, MI, 4‐OI, and HM4oRL groups, and C) the corresponding validation plot. D) Potential metabolic pathways for glucose, fatty acid β‐oxidation and tricarboxylic acid cycle derived from metabolomics of the infarcted myocardium. Glucose‐6P: glucose‐6‐phosphate; Fructose‐6P: fructose‐6‐phosphate; Acetyl‐CoA: acetyl‐coenzyme A; Succinyl‐CoA: succinyl‐coenzyme A; CPT1: carnitine palmitoyltransferase 1; CPT2: carnitine palmitoyltransferase 2; I‐IV: mitochondrial complex I‐IV; NADH: nicotinamide adenine dinucleotide. n = 6. E) Levels of FBA activity in injured myocardium after different treatments. n = 6. F) Levels of serum FFA after different treatments. n = 6. G‐H) Western blotting analysis of CPT2 protein content alterations in injured myocardium after different treatments. n = 4. I) The ROS production assessed by DHE staining of mice heart sections, and K) quantification of ROS levels in the infarcted area of each…

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  Figure 6. HM4oRL activated AMPK pathway signaling in vivo and in vitro. A) A Venn diagram intersecting the differentially expressed genes from transcriptomics with differentially expressed metabolites from metabolomics in MI versus HM4oRL groups. B) KEGG pathway enrichment analyses of 37 differentially expressed genes and differentially expressed metabolites. The AMPK and pho‐AMPK protein expression in C‐D) the infarcted myocardium, and E‐F) the hypoxia‐treated H9c2 cells were detected by Western blotting and quantified by ImageJ software. n = 3. Data are presented as mean ± SD. Statistical methods: One‐way ANOVA with Tukey's post‐test (D, F). In all panels, * indicates p < 0.05.

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  Figure 7. HM4oRL suppresses myocardial Ly6ChighCCR2highCX3CR1low monocyte infiltration and favors reparative CRM phenotypes after MI. A) Schematic illustration of the experimental timelines for short‐term studies of MI mice. B) Gating strategy for cardiac monocytes/macrophages and its subsets in mice hearts of each group after MI. C) Flow cytometry–based quantification of recruited monocytes CD64intLy6ChighCCR2highCX3CR1low in live CD45+ population. n = 4. D) Flow cytometry–based quantification of CD64highLy6ClowCCR2low monocytes/macrophages in live CD45+ population. n = 4. E) Flow cytometry–based quantification of MHC‐IIlowCX3CR1high cardiac‐resident macrophages in CD64highLy6ClowCCR2low monocytes/macrophages population. n = 4. H) Representative double‐immunofluorescence staining for F4/80 and CD206 in infarct tissue after MI, and F) quantification of CD206+ macrophages for each group by ImageJ software. Green, CD206; red, F4/80; blue, DAPI. n = 4. I) Representative double‐immunofluorescence staining for CD4 and FoxP3 in infarct tissue after MI, and G) quantification of CD4+ FoxP3+ cells for each group. Green, CD4; red, FoxP3; blue, DAPI. n = 5. Data are presented as mean ± SD.…

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  Figure 8. HM4oRL reduces infarct sizes and promotes cardiac repair after MI. A) Schematic illustration of the experimental timelines for short‐term studies of MI mice. B) Representative images of Masson's trichrome‐stained sections of infarcted hearts 7 days after treatment, and C) quantification of scar size for each group by ImageJ software. n = 6. Immunostaining analyses of D, H) collagen I (red), and E, I) collagen III (yellow) in border zone of infarct tissue. Blue, DAPI. n = 4. J) Representative double‐immunofluorescence staining for VEGF and CD68 in infarcted tissue after MI, and F) quantification of VEGF+ macrophages for each group by ImageJ software. Green, CD68; red, VEGF; blue, DAPI. n = 4. G, K) Immunostaining analyses of CD31 in the infarct zone. Red, CD31; blue, DAPI. n = 4.Data are presented as mean ± SD. Statistical methods: One‐way ANOVA with Tukey's post‐test (C‐G). In all panels, * indicates p < 0.05.

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  Figure 9. HM4oRL enhances long‐term cardiac function after MI. A) Schematic illustration of the experimental timelines for long‐term studies of MI mice. B) Representative M‐mode echocardiographic images of each group at 4 weeks post‐MI. Left ventricle EF C) and FS D) assessed by echocardiography at the indicated time points before and post MI for each group. n = 5. Quantification analysis for LVIDs E), LVIDd F), and LVPWs G) based on the M‐mode echocardiographic images at 4 weeks post‐MI. n = 5. Representative Sirius red staining images of myocardial sections I) and quantitative analysis of myocardial fibrosis size H) at 4 weeks post‐MI. n = 5. J) Representative immunofluorescence images of heart sections triple‐stained with fluorescein‐labeled α‐actinin (yellow), α‐SMA (red), CD31(green) and nuclei (blue) at 4 weeks post‐MI. Quantification of cardiomyocyte density (α‐actinin‐positive) K) and the number of vessels (both α‐SMA and CD31 positive) L) in the infarct zone. n = 5. M) Levels of FBA activity in injured myocardium after different treatments. n = 6. N) Levels of serum FFA after different treatments. n = 6. O‐P) Western blotting analysis of CPT2 protein content alterations in injured…