Functional evaluation of constructed pseudo-endogenous microRNA-targeted myocardial ultrasound nanobubble

Figures

• 

  Figure 1. General characteristics. (A) Clear NB lipid. (B) After mechanical vibration. (C) Microscopic image of NBs. (D) Connection of NBs with pAd-AAV-9/miRNA-1 gene complex by laser confocal microscope. (E) Schematic diagram of pAd-AAV-9/miRNA-1 targeted NBs. (F) Particle size distribution representative images of NBs and pAd-AAV-9/miRNA-1 NB by zeta analyzer. (G) Quantitative analysis of the diameter of NBs and pAd-AAV-9/miRNA-1 NB by zeta analyzer, data: mean ± SEM, *p < 0.05, the experiments were run three times, n = 3 per group. (H) Quantitative analysis of the concentration of NBs and pAd-AAV-9/miRNA-1 NB by zeta analyzer, data: mean ± SEM, *p < 0.05, the experiments were run three times, n = 3 per group. Stability of pAd-AAV-9/miRNA-1 NB by zeta analyzer, the changes in diameter (I) and concentration (J) of pAd-AAV-9/miRNA-1 NB at different time points.

• 

  Figure 2. Virus loading efficiency of pAd-AAV-9/miRNA-1 NB by Flow cytometry. (A) Representative image of pAd-AAV-9/miRNA-1 NB virus loading efficiency. (B) Quantitative analysis of the pAd-AAV-9/miRNA-1 NB virus loading rate, data: mean ± SEM, *p < 0.05 compared with the 1.25-μL group, #p < 0.05 compared with the 2.5-μL group, &p < 0.05 compared with the 5-μL group, the experiments were run three times, n = 3 per group.

• 

  Figure 3. Contrast-enhanced ultrasound imaging in vitro. (A) Preparation process diagram of ultrasonic imaging in an agarose gel matrix model. (B) Contrast-enhanced ultrasound imaging of SonoVue, NBs, and pAd-AAV-9/miRNA-1 NB in vitro. (C) Quantitative comparative analysis of the ultrasound contrast time-signal intensity curves between SonoVue, NB, and pAd-AAV-9/miRNA-1 NB groups. (D) Quantitative analysis of ultrasound contrast-enhanced imaging signal intensity difference in the NB group at different time points. (E) Quantitative analysis of ultrasound contrast-enhanced imaging signal intensity difference in the pAd-AAV-9/miRNA-1 NB group at different time points. Data: mean ± SEM, &p < 0.05 compared with the 5 s group, ♦p < 0.05 compared with the 15 s group, Φp < 0.05 compared with the 30 s group, #p < 0.05 compared with the 60 s group, the experiments were run three times, n = 3 per group.

• 

  Figure 4. Myocardial targeted contrast-enhanced ultrasound imaging in vivo. (A) Contrast-enhanced ultrasound imaging of the heart after injection of NBs or pAd-AAV-9/miRNA-1 NB at different time points. (B) Quantitative comparative analysis of the ultrasound contrast time-signal intensity curves between the NB and pAd-AAV-9/miRNA-1 NB groups. (C) Quantitative analysis of ultrasound contrast-enhanced signal imaging intensity difference in the NB group. (D) Quantitative analysis of ultrasound contrast-enhanced signal imaging intensity difference in the pAd-AAV-9/miRNA-1 NB group at different time points. Data: mean ± SEM, δp < 0.05 compared with the signal intensity of NBs after 1 min, Φp < 0.05 compared with the signal intensity of NBs after 5 min, θp < 0.05 compared with the signal intensity of NBs after 10 min, ϕp < 0.05 compared with the signal intensity of NBs after 15 min, *p < 0.05 compared with the signal intensity after 5 s, #p < 0.05 compared with the signal intensity after 15 s, &p < 0.05 compared the signal intensity after 30 s, §p < 0.05 compared with the signal intensity after 1 min, Δ p < 0.05 compared with the signal intensity after 5 min, the experiments were run three…

• 

  Figure 5. Biodistribution of NBs and pAd-AAV-9/miRNA-1 NB in SD rats by IVIS Imaging System. (A) Ex vivo fluorescence imaging of major organs of SD rats after the injection of NBs or pAd-AAV-9/miRNA-1 NB at different time points. (B) NB ROI analysis of fluorescence intensity of DiI uptake in different organs over time. (C) pAd-AAV-9/miRNA-1 NB ROI analysis of fluorescence intensity of DiI uptake in different organs over time. (D) Quantitative comparative analysis of the tendency of ROI fluorescence intensity between NBs and pAd-AAV-9/miRNA-1 NB in the heart after 5, 15, and 35 min. (E) Quantitative analysis of ROI fluorescence intensity of DiI uptake in the heart in the NB group after 5, 15, and 35 min. (F) Quantitative analysis of ROI fluorescence intensity of DiI uptake in the heart in the pAd-AAV-9/miRNA-1 NB group after 5, 15, and 35 min. Data: mean ± SEM, *p < 0.05 compared with the fluorescence intensity of NBs after 15 min, #p < 0.05 compared with the fluorescence intensity of NBs after 35 min, &p < 0.05, the experiments were run three times, n = 3 per group.

• 

  Figure 6. Fluorescence quantitative PCR validation of the expression levels of miRNA-1 in vital organs ex vivo. (A) Comparative analysis of the expression levels of miRNA-1in heart, kidney, spleen, lung, and liver of SD rats at different time points. (B) Analysis of the expression levels of miRNA-1 in different organs of SD rats over time. Data: mean ± SEM, ****p < 0.0001, the experiments were run three times, n = 3 per group.

• 

  Figure 7. Fluorescence quantitative PCR validation of the expression levels of miRNA-1 of different groups in heart ex vivo. Data: mean ± SEM, &p < 0.05 compared with the pAd-AAV-9/miRNA-1 NB group, *p < 0.05 compared with the pAd-AVV-9/miRNA-1 NB + UTMD group, the experiments were run three times, n = 3 per group.

• 

  Figure 8. Cytotoxic analysis of pAd-AAV-9/miRNA-1 NB in vitro.