Ultrasound targeted fatty acid nitroalkene-containing lipid nanoparticles attenuate inflammation and reverse myocardial fibrosis

Figures

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  Figure 1. Targeted delivery of NO2-FA by UTC+NO2-FA LNPs to the target sites (heart and hindlimb) in rodent and porcine models. A Fabrication of NO2-FA LNPs from lipids and NO2-FA. The PFB gas entrapped in the core enables the LNPs to cavitate when US is applied. B Experimental setup for the targeted delivery of NO2-FA to healthy hindlimb of rat using intrajugular venous infusion of NO2-FA LNPs and simultaneous application of external focused ultrasound over the target hindlimb. C Quantification of NO2-FA in gastrocnemius (treatment site) and quadriceps femoris (remote site) after UTC+NO2-FA LNP or IV NO2-FA administration. Data are mean ± SEM of 6 rats per group; one-way ANOVA with Tukey’s multiple comparisons. D Experimental setup for the targeted delivery of NO2-FA to rat heart after inducing ischemia reperfusion injury, using intrajugular venous infusion of NO2-FA LNPs and simultaneous application of external focused ultrasound over the heart. E Quantification of NO2-FA in myocardial tissue (treatment site) and quadriceps femoris (remote site) after UTC+NO2-FA LNP or IV NO2-FA administration. Data are mean ± SEM of 4 rats per group; one-way ANOVA with Tukey’s multiple comparisons. F…

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  Figure 2. UTC+NO2-FA LNPs significantly improve key cardiac function parameters post Ischemia Reperfusion Injury (IRI). A Flow chart showing important steps of IRI experiment. B Representative M-mode ultrasound images of rat heart showing the movement of myocardium, recorded at 2 stages i.e. during ischemia and 2h post reperfusion from 4 groups (sham, control, IV NO2-FA only and UTC+ NO2-FA LNPs). Each image contains important information including ejection fraction (EF) %, fractional shortening (FR) %, internal diameter, heart rate (HR), stroke volume (SV), cardiac output (CO) and more. C Delta (Δ) EF % from during ischemia to 2h post reperfusion for control, IV NO2-FA only and UTC+NO2-FA LNPs groups. Data are mean ± SEM from 6 rats per group D Delta (Δ) FS % from during ischemia to 2h post reperfusion for control, IV NO2-FA only and UTC+NO2-FA LNPs groups. Data are mean ± SEM from 5-6 rats per group. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons test.

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  Figure 3. UTC+NO2-FA LNPs reduce infarct size, ameliorate inflammation and myocardial injury post Ischemia Reperfusion Injury (IRI). A Representative transverse section images of rat heart stained with Masson's trichrome. Area in the yellow box in each representative image is magnified at 12-15× below for better visualization. B–E Changes in mRNA expression of key inflammatory genes, including IL-6, MCP-1, NF-κB, and TNF-α, in myocardial tissue samples collected 2 h post-ischemia-reperfusion injury (IRI) from Sham, Control, IV NO₂-FA only, and UTC+NO₂-FA LNP groups (n = 4 per group). F–I Serum levels of myocardial injury and inflammation biomarkers, including cardiac troponin, creatinine phosphokinase, myoglobin, and lactate dehydrogenase, measured 2h post-IRI in Sham, Control, IV NO₂-FA only, and UTC+NO₂-FA LNP groups (n = 5–6 per group). All data are presented as mean ± SEM. Statistical significance in panels B–I was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Figure 4. UTC+NO2-FA LNPs significantly improve key cardiac function parameters post fibrosis. A Flow chart showing important steps in the development of fibrosis. B Experimental setup for the treatment of myocardial fibrosis with IV NO2-FA and UTC+NO2-FA LNPs. C Development of rat myocardial fibrosis model. Transverse section images of rats (4) heart stained with Masson's trichrome taken at 28 days post IRI. Area in the black box in each representative image is magnified at 12-15× below for better visualization. D Flow chart showing important steps and timelines in the development of fibrosis and its treatment. E Representative M-mode ultrasound images of rat heart showing the movement of myocardium, recorded at 2 stages i.e. fibrosis, pre-treatment and post treatment. F Left ventricular (LV) fractional area change (%) measured at two stages-fibrosis (pre-treatment) and post-treatment in Sham, Control, IV NO₂-FA only, and UTC+NO₂-FA LNP groups (n = 7-12 per group). G Left ventricular wall motion score index (WMSI) measured at two stages-fibrosis (pre-treatment) and post-treatment in Sham, Control, IV NO₂-FA only, and UTC+NO₂-FA LNP groups (n = 7-12 per group). All data are presented as…

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  Figure 5. UTC+NO2-FA LNPs reverse fibrosis and reduce neutrophil infiltration. A Representative transverse section images of rat heart stained with Masson's trichrome. Area in the yellow box in each representative image is magnified at 12-15× below for better visualization. B Representative transverse section images of rat heart stained with H&E. Area in the yellow box in each representative image is magnified at 12-15× below for better visualization. C Cumulative myocardial fibrosis (%) in cardiac sections post-treatment (n = 3-7 per group). D Cumulative neutrophil infiltration (%) in cardiac sections post-treatment (n = 3–7 per group). All data in C-D are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Figure 6. UTC+NO2-FA LNPs significantly reduced fibrotic lesions as revealed by Immunohistochemistry (IHC). A-C Representative transverse section images of rat heart stained with collagen 3 A, collagen 1 B and α-smooth muscle actin (SMA) C antibodies. Area in the red box in each representative image is magnified at 12-15× below for better visualization. D Cumulative myocardial collagen 3 (%) in the section post treatment (n = 3). E Cumulative myocardial collagen 1 (%) in the section post treatment (n = 3-4). F Cumulative myocardial α-SMA (%) in the section post treatment (n = 3-4). All data in D–F are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Figure 7. UTC+NO2-FA LNPs reduce infarct and ameliorate inflammation post fibrosis. A Representative transverse section images of rat heart stained with Evan’s blue and TTC. Area in the yellow box in each representative image shows the infarct area. B Cumulative left ventricular (LV) infarct area (%) in cardiac sections post-treatment (n = 3 per group). C-E Serum levels of key biomarkers of myocardial injury, fibrosis, and inflammation measured 14 days post-treatment in Sham, Control, IV NO₂-FA only, and UTC+NO₂-FA LNP groups. C Creatinine phosphokinase (n = 6-7 per group). D Lactate dehydrogenase (n = 6 per group). E Myoglobin (n = 6 per group). All data in B-E are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Figure 8. UTC+NO2-FA LNPs treatment reduced LGE and ECV (%), enhanced cardiac output and improved longitudinal strain post fibrosis. A Representative LGE images of the heart. Bright white areas in the myocardium show fibrosis. B Representative ECV images of the heart. Blue color in the circle shows less ECV while red patches show high ECV. C Cumulative cardiac output (mean ± SEM), calculated as stroke volume × heart rate, for control, IV NO₂-FA, and UTC+NO₂-FA LNPs groups (n = 3). D Cumulative left ventricular ejection fraction (LV EF, mean ± SEM) at the end of the experiment (n = 2-5). E Cumulative longitudinal strain (mean ± SEM) at the end of the experiment (n = 2-4). F Cumulative extracellular volume (ECV, %, mean ± SEM) measured prior to euthanasia (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.