Treating myocardial infarction via a nano-ultrasonic contrast agent-mediated high-efficiency drug delivery system targeting macrophages

Figures

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  Fig. 1.. Graphical abstract of US-responsive nanoparticles. Synthesis process of OA-si@FA-PNBs and a schematic illustration of their effects on improving MI in mice under US assistance.

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  Fig. 2.. Characterization and performance of FA-PNBs loaded with siRNA and nitro-fatty acids: Stability, imaging, and transfection efficiency. (A) TEM image. Scale bar, 100 nm. (B) Size distribution analysis of FA-PNBs and OA-si@FA-PNBs. (C) Stability of the OA-si@FA-PNBs. (D) Surface zeta potential of the FA-PNBs, si@FA-PNBs, OA@FA-PNBs, OA-si@FA-PNBs, and si-Cy5@FA-PNBs. (E) Agarose gel electrophoresis of FA-PNBs loaded with siSTAT1. (F) Hemolysis experiment. Equal volumes of PBS, FA-PNBs, si@FA-PNBs, OA@FA-PNBs, OA-si@FA-PNBs, and ddH2O were added to the blood cells of the mice. (G) Confocal images of Cy5-tagged siSTAT1 under different transfection conditions. Red indicates Cy5-conjugated siRNA (siSTAT1-Cy5). Scale bar, 50 μm. (H) Flow cytometry analysis of Cy5-tagged siSTAT1 under various conditions. (I) Statistical analysis of the flow cytometry results. (J) Confocal imaging of DiO-labeled cell membranes (green) and Cy5-conjugated OA-si@FA-PNBs (red) under different conditions. Scale bar, 25 μm. siSTAT1, siRNA targeting STAT1. The data are expressed as the means ± SDs. n = 3 per group. *P < 0.05 and ***P < 0.001. ns, not significant.

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  Fig. 3.. FA-modified PNBs (OA-si-Cy5@FA-PNBs) were notably enriched in the MI area. US can extend the enrichment time of these materials. (A) Live optical imaging of sham and MI model mice 2 hours after FA-PNB or PNB injection. Sham + FA (+) indicates sham mice that received tail vein injections of PNBs with FA modification. MI + FA (−) indicates MI mice that received tail vein injections of PNBs without FA modification. MI + FA (+) indicates MI mice that received tail vein injections of FA-PNBs. (B) Quantification of fluorescence intensity in the precordial area of the heart. n = 4 per group. (C) Ex vivo optical imaging of infarcted hearts from sham and MI model mice 2 hours postinjection. (D) Statistical analysis of the average fluorescence intensity in ex vivo hearts. n = 6 per group. (E) Live optical imaging after intravenous injection of FA-PNBs with or without external US treatment at 0, 2, 48, and 72 hours. MI + US (+) indicates MI mice that were irradiated with US for 3 min after tail vein injection. (F) Quantitative analysis of fluorescence intensity in the precordial area of the heart. n = 3 per group. FRβ, FA receptor β; FA, folic acid. The data are expressed as the means ±…

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  Fig. 4.. Precision codelivery of siSTAT1 and OA-NO2 to CD86-positive macrophages in MI using FA-modified nanoparticles. (A) Flow cytometry analysis of FRβ-positive and CD86-positive cells in the anterior wall of the left ventricle of MI mice on day 3. (B) Proportion of CD86-positive cells among FRβ-positive cells was 95.23%, as determined by flow cytometry. n = 3 per group. (C) Flow cytometry analysis of CD86-positive cells among TRITC+/Cy5+ cells in the MI area. siSTAT1 was labeled with Cy5, OA-NO2 was labeled with TRITC, and OA-TRITC-siSTAT1-Cy5@FA-PNBs were administered via tail vein injection 3 days post-MI. (D) Quantification of the flow cytometry data revealed that 97.6% of the TRITC+/Cy5+ cells were CD86 positive. n = 7 per group. (E) Confocal microscopy detection of Cy5, TRITC, and CD86 triple-positive cells in the ischemic region. The arrows indicate triple-positive CD86, OA-TRITC, and siSTAT1-Cy5 cells. Scale bar, 50 μm. (F) Quantitative analysis of Cy5-positive, TRITC-positive, CD86-positive, and triple-positive cells in the ischemic region. n = 4 per group. (G) Flow cytometry assessment of Cy5 and TRITC double-positive, Cy5-only, TRITC-only, and double-negative cells in the…

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  Fig. 5.. Transcriptome analysis of macrophages treated with different FA-PNBs. (A) Gene Ontology (GO) analysis highlighting the biological processes in CD86-positive macrophages treated with OA@FA-PNBs versus NC@FA-PNBs. (B) KEGG pathway analysis of the DEGs. (C) Heatmap of inflammation-related gene expression changes in the various groups. (D) Quantitative differential gene expression. (E to J) qRT-PCR quantification of macrophage-related gene expression levels. Green, NC@FA-PNBs group; yellow, OA@FA-PNBs group; blue, si@FA-PNBs group; purple, OA-si@FA-PNBs group. US, ultrasound; MI, myocardial infarction; FA-PNBs, folic acid-perfluorohexane nanobubble system; PNBs, perfluorohexane nanobubble system; siSTAT1, siRNA targeting STAT1; OA-NO2, nitric acid; FA, folic acid. The data are presented as the means ± SDs, n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

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  Fig. 6.. OA-si@FA-PNBs modulated the macrophage phenotype and reduced inflammation in vitro. Twenty-four hours after LPS induction, CD86-positive macrophages were treated with NC@FA-PNBs, OA@FA-PNBs, si@FA-PNBs, or OA-si@FA-PNBs and subjected to ultrasonic stimulation for 3 min. The results were analyzed 3 days after treatment. (A) Schematic showing the procedures used for macrophage polarization and phenotype detection. (B) Proportion of CD86-positive cells was detected by flow cytometry. (C) Statistical map showing the proportion of cells positive for CD86. (D) Proportion of Cx3cr1-positive cells was detected by flow cytometry. (E) Statistical map showing the proportion of Cx3cr1-positive cells. (F) Scanning confocal microscopy detection of CD86 (green), CD11b (red) and DAPI (blue) expression. Scale bar, 50 μm. The arrows indicate cells that are double positive for both CD86 and CD11b. (G and H) CD86-positive macrophage markers IL-1β and TNF-α were detected via ELISA. (I and J) ELISA detection of IL-4 and TGF-β1. Green, NC@FA-PNBs group; yellow:,OA@FA-PNBs group; blue, si@FA-PNBs group; purple, OA-si@FA-PNBs group. The data are expressed as the means ± SDs. n = 3 per group. *P < 0.05,…

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  Fig. 7.. OA-si@FA-PNBs improve the post-MI immune microenvironment in mice. (A) Schematic showing the procedures of evaluating immune microenvironment and cardiac function in MI mice. (B) Flow cytometry analysis and the quantification (C) of the proportion of CD86-positive macrophages in the ischemic myocardial injury region of MI mice under different treatments. n = 4 per group. (D) Flow cytometry analysis and the quantification (E) of the proportion of Cx3cr1-positive macrophages in the ischemic region of MI mice under different treatments. (NC@FA-PNBs group, n = 5; OA@FA-PNBs groups, n = 4; si@FA-PNBs group, n = 3; OA-si@FA-PNBs group, n = 6). (F) Flow cytometry analysis and the Quantification (G) of the proportion of Tregs (CD4+/FOXP3+ cells) in the ischemic region of MI mice under different treatments. (NC@FA-PNBs group, n = 6; OA@FA-PNBs groups, n = 4; si@FA-PNBs group, n = 6; OA-si@FA-PNBs group, n = 6). (H) Immunostaining of MI mice on day 7 post-MI for the CD86 (green) and Cx3cr1 (red) in the ischemic border zones. (I) Immunostaining of MI mice on day 7 post-MI for the Cx3cr1 (red) and Ki67 (green) the ischemic border zones. The arrows indicate cells that are double positive for…

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  Fig. 8.. OA-si@FA-PNBs preserve cardiac function in MI model mice. (A) Echocardiography of the LV long-axis end diastolic/systolic volume in the treatment and sham groups 28 days post-MI. (B and C) Assessment of the LV long-axis EF and FS before MI in the treatment and MI-only groups at 7 and 28 days post-MI (sham group, n = 6; NC@FA-PNBs group, n = 6; OA@FA-PNBs group, n = 6; siSTAT1@FA-PNBs group, n = 6; OA-si@FA-PNBs group, n = 10). (D) Peak of the mitral early (E)/late (A) ventricular filling velocity in the treatment and MI-only groups 28 days post-MI. (E) Quantitative analysis of the mitral E/A peak ratio (sham group, n = 6; NC@FA-PNBs group, n = 6; OA@FA-PNBs groups, n = 6; si@FA-PNBs group, n = 6; OA-si@FA-PNBs group, n = 10). (F) Heart masson staining of the treatment and MI-only groups on day 28 post-MI: fibrosis area (blue) and myocytes (red). (G) Quantitative analysis of the LV wall thickness in 12 segments individually. (H) Quantitative analysis of infarct zone fibrosis length and (I to K) quantitative analysis of the LV wall thickness in the septal zone, infarct zone and border wall (sham group, n = 6; NC@FA-PNBs group, n = 6; OA@FA-PNBs group, n = 6; si@FA-PNBs group, n =…