Adaptive bioactivable nanosystems for synergistic myocardial infarction therapy using traditional pharmaceutics

Figures

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  Scheme 1. Development of an inflammation-activated size variable NP TF-5NP to achieve effective long-term retention of TIIA and FM.

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  Fig. 1. Inflammation-activated size variable and cTnT anchored NPs. (A) Schematic diagram of MPO converting the phenolic residues of DSPE–PEG–bis-5HT to radicals, enabling the bis-5HT radicals to form bonds. (B) TEM images of TF-5NP in PBS or PBS catalyzed by MPO in the presence of H2O2, scale bar: 100 nm. (C) Zeta potentials of TF-NP and TF-5NP. (D) Size distributions of TF-NP and TF-5 NP under different conditions: PBS alone, MPO/H2O2 activation, and neutrophil treatment. (E) Schematic diagram of MPO converting the phenolic residues of DSPE–PEG–bis-5HT and bis-5HT radicals, anchoring to the cysteine residue of cardiac cTnT. (F) Confocal fluorescence images of MI tissue slices after intravenous administration of cy5.5, cy5.5-NP, and cy5.5-5-NP for 14 d. Blue channel: nucleus; green channel: cTnT; red channel: cy5.5-labeled MPO-responsive NPs (cy5.5-5 NP) or cy5.5-labeled nonMPO-responsive NPs (cy5.5-NP) or cy5.5. Scale bar: 50 μm. (G) Release profiles of TIIA from TF-5 NP under different conditions: untreated, +MPO, +H2O2, +MPO/H2O2, +MPO/H2O2 + 4-ABAH (MPO inhibitor), and +neutrophils. All data are presented as mean ± SD (n = 3). Statistical significance was determined via one-way…

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  Fig. 2. Evaluation of cell activity and apoptotic in vitro. (A) Relative cell viability of HUVECs in the anoxic environment after 48 h of incubation with TIIA, FM, TIIA + FM, TF-NP, and TF-5NP. Annexin-V/PI apoptosis assay of HUVECs treated with each group for 48 h measured via (B) FCM and (C) statistics analyses. (D) Representative fluorescence micrographs showcasing the expression of Ki67 (green) in NRCMs, with sarcomeric α-actinin (α-SA) labeling (red). Scale bar: 100 μm. (E) Quantification of Ki67+ cells using ImageJ. (F) Representative fluorescence micrographs from the LIVE/DEAD assay assessing the viability of NRCMs. Scale bar: 100 μm. (G) NRCM viability measured using ImageJ. (H) Representative fluorescence micrographs of cell apoptosis detected using the TUNEL assay. Scale bar: 100 μm. (I) Percentage of TUNEL + NRCM determined using ImageJ. Western blotting (J) and quantification (K) of cleaved Caspase-3, Bax, and Bcl-2. All data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined by one-way ANOVA. ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Fig. 3. Effects of TF-5NP on angiogenesis and mitochondrial function in vitro. (A) Experimental diagram of TF-5NP intervention in HUVEC migration and tubule formation. (B) Images of HUVEC migration. scale bar: 200 μm. (C) Quantitative analysis of migration rates for 12 and 24 h. (D) Tube formation assay of HUVECs. Scale bar: 200 μm. (E) Quantitative analysis of the number of junctions. (F) Western blotting and quantification analysis of (G) VEGF, (H) PDGF, and (I) FGF expression levels. (J) Quantitative analysis of the ATP level. (K) Evaluation of mitochondrial membrane potential using MitoTracker staining. Scale bar: 100 μm. (L) Evaluation of intracellular ROS levels using MitoSOX staining. Scale bar: 100 μm. (M) Mitochondrial membrane potential levels were evaluated by Mito-JC-1 staining. Scale bar: 100 μm. All data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined via one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Fig. 4. TF-5 NP protects against cardiac dysfunction and effect on angiogenesis in zebrafish. (A) Schematic diagram of Verapamil-induced cardiac dysfunction zebrafish administration and detection. (B) Photomicrographs of zebrafish with red and yellow-dotted lines marking the pericardial sac and areas of venous congestion. Scale bar: 200 μm. (C) Statistical analysis of pericardial sac area in zebrafish. (D) Statistical analysis of venous congestion area in zebrafish. (E) Representative images of zebrafish hearts showing verapamil-induced heart dilation, which was mitigated by treatment with TF-5 NP. Scale bar: 200 μm. (F) Quantification of cardiomyocyte area in zebrafish across each experimental. (G) Measurement of the distance between sinus venosus and bulbus arteriosus (SV-BA distance), in zebrafish across each group. (H) Analysis of blood flow velocity in zebrafish. (I) Heart rates recording and statistical analysis in each group. (J) Cardiac output of zebrafish in each experimental group was determined and subjected to statistical analysis. (K) The name of each blood vessel of zebrafish is marked with schematic diagram and fluorescence images utilized to observe the structural…

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  Fig. 5. Effects of TF-5 NP on pharmacokinetics in pigs. (A) Pigs were intravenouslyinjected with cy5.5 and cy5.5-NP, cy5.5-5 NP with the same dosage of cy5.5 (2 mg kg-1). (B) The blood samples were collected at predetermined intervals and the fluorescence intensity was subsequently measured by in vivo imaging system. (C) Quantitative analysis of fluorescence intensity. Histological sections of the infarct region were immunolabeled with the (D) Endothelial marker CD31 (green), (F) Cardiomyocytes marker α-SMA (green), (H) Neutrophil marker CD11b (green), (J) MPO (green), cy5.5 (white) and DAPI (blue). Scale bar: 100 μm. Quantitative analysis of (E) CD31, (G) α-SMA, (I) CD11b and (K) MPO fluorescence intensity was conducted using Image J. All data are presented as mean ± SD (n = 3). Statistical significance was determined via one-way ANOVA. ∗∗∗P < 0.001.

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  Fig. 6. TF-5 NP attenuated adverse LV remodeling in pigs. (A) MR imaging and heart sectioning were performed on pigs on day 28. (B) Representative images of hearts harvested 28 days post-MI, with and without TF-5 NP treatment. Representative histological analysis of the infarcted myocardium was performed among different treatment groups using (C) H&E staining and (D) Masson's trichrome staining. (scale bars: 3 mm in overall tissue image, 200 μm in low-magnification images, and 50 μm in high-magnification images). Quantitative analysis of the (E) Percentage of Infarction area heart area, and (F) Percentage of collagen fiber area. (G) Representative images showcasing WGA immunofluorescence staining. Scale bar: 50 μm. (H) Representative MR images in short (upper pannel) and long (bottom) axis views at the area of the infarct scar at 28 days post-MI. The infarct area appears in white (circled), while the non-infarcted myocardium appears black. (I) Representative MR images in Systole and diastole. Quantitative analysis of (J) Left Ventricular End Systolic Volume, (K) left ventricular end-diastolic volume, (L) Body surface area, (M) indexed left ventricular end-systolic volumes, (N) indexed…

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  Fig. 7. Mechanism underlying the effects of TF-5 NP on regulate the inflammation and promoting angiogenesis. (A) The volcano plot illustrates the discovery of differentially expressed transcripts (DETs) using a significance threshold of P < 0.05 and a fold change (FC) of ≥5 or ≤0.2. (B) Venn diagrams from DETs analysisidentified 365 transcripts of interest. (C) A heatmap depicting selected proteins that represent significantly altered signaling pathways. (D) Principal component analysis between sham, model and TF-5 NP group. (E) Gene Ontology (GO) enrichment results. (F) Tanshinone IIA inhibits ventricular remolding after MI. (G) Representative images showcasing TUNEL staining. Scale bar: 0.5 μm. (H) Western blot of TOM20 Flotillin-1 and TOM40 expression levels. (I) Histological sections of the infarct region and border region were immunolabeled with the pyroptosis specific protein, Caspase-1, GSDMD, IL-1β, IL-4 (orange), DAPI (blue). Scale bar: 20 μm. (J) Schematic diagram of Formononetin regulating angiogenesis through apoptosis. (K) Representative electron microscopic picture of mitochondria. scale bars: 50 μm in low-magnification images, and 20 μm in high-magnification images. (L)…