Targeted neutrophil-mimetic liposomes promote cardiac repair by adsorbing proinflammatory cytokines and regulating the immune microenvironment

Figures

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  Fig. 1. Fabrication of Neu-LPs. A–C DLS measurements of Neu-LP size (A), zeta potential (B), and polydispersity index (C) (n = 3). D Representative TEM image of Neu-LPs stained with uranyl acetate. Scale bars, 50 nm. E Confocal fluorescence microscopy images and co-localization analysis of Neu-LPs (top) and a physical mixture of LPs and NMVs (bottom). Green, NMVs; red, LPs; scale bars, 500 nm. Statistical analysis was performed using Student’s t-test. Data are presented as mean ± SD

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  Fig. 2. Membrane protein composition of Neu-LPs. A SDS-PAGE analysis of neutrophils, NMVs, and Neu-LPs. B Western blotting analysis of characteristic protein bands (TNFαR, IL1βR, IL6R, LFA-R, and CXCR2) in NMVs and Neu-LPs. NLWNM: Neutrophil lysate without neutrophil membrane protein

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  Fig. 3. Inflammatory cytokine binding ability of Neu-LPs in vitro. A Neutralization dose response of proinflammatory factors (TNFα, IL1β, IL6, and CXCL2) by Neu-LPs. *P < 0.05 and ***P < 0.001 compared with the 0 mg/ml group. B Binding capacity of Neu-LPs to inflammatory cytokines (TNFα, IL1β, IL6, and CXCL2). IC50 values were derived from the variable slope model using GraphPad Prism 7. C Schematic illustration of a direct co-culture of macrophages and cardiomyocytes. D Expression levels of proinflammatory cytokines (TNFα, IL1β, IL6, and CXCL2) in the in vitro myocardial inflammation model with or without Neu-LP treatment. Statistical analysis was performed using one-way ANOVA. Data are presented as mean ± SD. **P < 0.01 and ***P < 0.001 compared with the NC group

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  Fig. 4. Cardioprotective and immunoregulatory effects of Neu-LPs in vitro. A Representative TUNEL staining of inflammation-stimulated cardiomyocytes treated with or without Neu-LPs. B Quantitative PCR analysis of apoptosis genes in cardiomyocytes after treatment with Neu-LPs (n = 3). C, D Microscopy images (C) and quantitative analysis (D) of neutrophil chemotaxis after treatment with Neu-LPs (n = 3). E Expression levels of M1 and M2 macrophage markers following treatment with Neu-LPs. F, G Representative flow cytometry graphs (F) and quantitative analysis (G) of macrophage polarization after treatment with Neu-LPs (n = 3). H Representative fluorescence images and quantitative analysis of an EdU assay for endothelial cell proliferation rate (n = 3). Scale bars, 50 μm. I Quantitative PCR of proliferation genes in HUVECs after treatment with Neu-LPs (n = 3). J Representative fluorescence images and quantitative analysis of tube formation by HUVECs after treatment with Neu-LPs (n = 4). Scale bars, 100 μm. Statistical analysis was performed using one-way ANOVA. Data are presented as mean ± SD. NC, negative control group which is treated with PBS. *P < 0.05, **P < 0.01, and ***P < 0.001…

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  Fig. 5. Biodistribution of Neu-LPs in vitro and in vivo. A Schematic illustration of the in vitro transmembrane model to evaluate the penetration and transport of Neu-LPs across a monolayer of HUVECs. B Quantification of Neu-LP distribution in the transwell system (n = 3). C, D Ex vivo fluorescence images (C) and quantification (D) of hearts from MI/R mice after treatment with Neu-LPs, LPs, or PBS at various timepoints (3 h, 1 day, and 3 days) (n = 6). *P < 0.05 and ***P < 0.001 compared with the PBS group. E Fluorescence microscopy images of Neu-LP distribution in infarcted areas. Scale bars, 100 μm. F Quantitative analysis of the fluorescence intensity of Neu-LPs (n = 6). ***P < 0.001 compared with the LPs group. G, H Representative fluorescence images (G) and quantitative analysis of fluorescence intensity (H) in major organs after intravenous injection of Neu-LPs (n = 6). *P < 0.05 and **P < 0.01 compared with the PBS group. Comparisons between two groups were performed with Student’s t-test. One-way analysis of variance (ANOVA) was employed for comparisons among more than two groups. Data are presented as mean ± SD

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  Fig. 6. In vivo immunoregulation ability of Neu-LPs. A Concentration profiles of inflammatory cytokines (TNFα, IL1βand IL6) and chemokine (CXCL2) in ischemic hearts at various timepoints (1, 3, and 7 days) following injection of Neu-LPs (n = 3). B Infiltration of neutrophils in the infarcted area three days after Neu-LP treatment detected by myeloperoxidase (MPO) (n = 3). Scale bars, 100 μm. C Quantitative analysis of MPO-positive cells. D, E Fluorescence images (D) and quantitation (E) of macrophages (F4/80, red) and M2 macrophages (CD206, green) in heart tissue (n = 3) three days after Neu-LP treatment. Scale bars, 100 μm. Statistical analysis was performed using one-way ANOVA. Data are presented as mean ± SD. *P < 0.05 and **P < 0.01 compared with the PBS group

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  Fig. 7. Cardiac repair effects of Neu-LPs in vivo. A Representative Masson’s trichrome staining of the base, mid-left ventricular (LV), and apical regions of an infarcted heart 4 weeks after treatment with Neu-LPs. Scale bars, 2.5 mm. B LVEF, FS, LVEDV, and LVESV values measured by echocardiography at 4 weeks (n = 6 animals per group). C Representative TUNEL staining images and quantification of TUNEL-positive apoptotic cells in infarcted regions 7 days after MI (n = 3). Scale bars, 100 μm. D Representative confocal images and quantitative analysis of PH3-positive nuclei in the peri-infarct region 4 weeks after treatment with Neu-LPs (n = 3). Scale bars, 100 μm. E Representative images and quantification of Ki67-positive cells at 7 days post-treatment with Neu-LPs (n = 3). Scale bars, 100 μm. F Representative micrographs of CD31-positive blood vessels and quantification of capillary density in hearts 4 weeks after treatment with Neu-LPs (n = 3). Scale bars, 100 μm. Statistical analysis was performed using one-way ANOVA. Data are presented as mean ± SD. *P < 0.05 and **P < 0.01 compared with the PBS group

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  Fig. 8. Safety evaluation of Neu-LPs. A H&E staining of the major organs (n = 6). Scale bars, 50 μm. B, C Quantitative analysis of blood biochemistry results indicating liver (B) and kidney (C) function from peripheral blood samples. Statistical analysis was performed using one-way ANOVA. Data are presented as mean ± SD