Fig 1.
Characterization of microbubbles (MBs).
(A) Schematic representation of cationic lipid MBs. The lipid composition of the MBs included DPPC, DSPE-PEG2000, DSTAP, and DC-Chol. DSPE-PEG2000 was utilized to improve the stability of MBs, while DSTAP and DC-Chol were incorporated to positively charge the surface and facilitate the binding of eNOS-siRNA through charge interactions. Diagrams were generated in BioRender (https://biorender.com; 2024). (B) Representative confocal image of siRNA-loaded MBs (siRNA-MBs). (C) The histogram represents the size distribution of cationic unloaded MBs and siRNA-loaded cationic MBs (siRNA-MBs). Three independent MBs preparations were analyzed. (D) Direct binding of siRNA to cationic MBs was visually confirmed by confocal microscopy. Cationic MBs loaded with the lipophilic membrane marker Vybrant Dil are observed as red fluorescence (a). 6-FAM-labeled siRNA is depicted as green fluorescence (b). Merge of red and green channels depicted as yellow fluorescence (c), showed colocalization of siRNA and the phospholipid monolayer of the cationic MBs. Neither binding of siRNA (f) or colocalization (g) was observed in neutral MBs. (E) Quantitative assessment of siRNA binding to cationic MBs. The siRNA loading capacity of cationic MBs was higher compared to neutral MBs as determined by the curve plateau. (F) Quantitative assessment of siRNA-loading efficiency. Data represent the mean ± standard deviation for three different experiments. (E) *P ≤ 0.0001 vs neutral MBs. (F) ** P ≤ 0.05 vs 10 μg.
Fig 2.
Monolayer integrity and cell viability following exposure to UMMD.
(A) Representative fluorescence images of the endothelial monolayer exposed to US (0.50 W/cm2 of US intensity) using different MBs/cell ratios: (a) untreated cells, (b) cells exposed only to US, (c), (d), (e), (f), and (g) correspond to 1:1, 4:1, 6:1, 8:1, or 12:1 MBs/cell ratios, respectively. (B) Endothelial monolayer integrity and (C) cell viability were evaluated after UMMD treatment at 0.50, 1, and 2 W/cm2 US intensities using 1:1, 4:1, 6:1, 8:1, or 12:1 MBs/cell ratios. (D) Monolayer integrity and (E) cell viability at 0 and 24 h after UMMD treatment with a 6:1 MBs:cell ratio, 10% of duty cycle, and 2 W/cm2 of intensity. Data represent the mean of percentage ± standard error of the mean for three different experiments (n = 3). *P ≤ 0.05 vs control, **P ≤ 0.05 vs 0 h.
Fig 3.
Delivery of 6-FAM-labeled siRNA assisted by ultrasound-mediated microbubble destruction (UMMD).
(A) Confocal images revealed the intracellular uptake of 6-FAM siRNA (green) of UMMD-treated bEnd.3 cells as a function of time. Nuclei were stained with Hoechst 3342 (blue). Control without treatment after 6, 12 or 24 h (a, b, and c, respectively). Cells transfected using lipofectamine and 6-FAM siRNA at 6, 12 or 24 h after treatment (d, e, and f, respectively). Cells incubated with 6-FAM siRNA-MBs exposed to US at 6, 12 or 24 h after treatment (g, h, and i, respectively). (B) Fluorescence quantification of 6-FAM siRNA uptake. Data represent the mean ± standard error of the mean for three different experiments. *P ≤ 0.05 vs lipofectamine + NC-siRNA.
Fig 4.
Comparison of eNOS-siRNA transfection delivered by UMMD and using lipofectamine on mRNA and protein expression.
(A) Measurement of eNOS mRNA expression levels in UMMD-transfected cells using eNOS-siRNA-loaded MBs and 0.25, 0.50, 1, or 2 W/cm2 US intensities. (B) eNOS mRNA expression levels were evaluated by qPCR after 24 h of UMMD-transfection and lipofection. (C) eNOS protein expression levels were assessed by Western blotting 24 h post-transfection. Beta-actin was used as loading control. Data represent the mean ± standard error of the mean for three different experiments. (A and C) *P ≤ 0.05 vs control. (B) *P ≤ 0.05 vs lipofection 42 ng.
Fig 5.
Transfection of eNOS siRNA by MBs and UMMD efficiently inhibits NO production in bEnd.3 cells.
The upper panel (A) shows, representative confocal images depict endothelial NO release under different conditions: (a) Basal NO release; (b) Acetylcholine (Ach, 100 μM) stimulation; (c) Ach stimulation with NOS inhibitor L-NAME (1 mM); (e) Ach stimulation after transfection with eNOS-siRNA (42 ng)-MBs exposed to ultrasound (US); and (g) Ach stimulation after transfection with eNOS-siRNA (79 ng) using lipofectamine. NC-siRNA was used as a control for both UMMD and lipofectamine transfection (d and f, respectively). Quantification of fluorescence intensity (arbitrary units) of cells with or without Ach stimulation or treatment (control) is shown in panel (B). Each experimental condition was conducted in duplicate, and the entire experiment was replicated three times (n = 3). Data represent the mean ± standard error of the mean for three different experiments. *P ≤ 0.05 vs Ach.
Fig 6.
Effective inhibition of ex vivo angiogenesis mediated by eNOS-siRNA transfection using UMMD.
The aortic ring assay was used as ex vivo model to evaluate angiogenesis in rats. (A) The image of an aortic ring at day 0 reveals the absence of microvessel sprouts. By day 6, EC proliferation becomes evident. (B) Characterization of the angiogenic response with respect to siRNA concentration. (C) Phase-contrast images of rat VEGF-treated aortic rings grown in type I collagen. (a) Control aortic rings, (b) aortic rings transfected using lipofectamine and 131 ng eNOS-siRNA, and (c) aortic rings treated with eNOS-siRNA loaded-MBs (131 ng) and exposed to US. (d, e, and f) corresponding processed images, respectively, showing in white the formation of new vessels. (D) Quantification of angiogenesis response using image analysis. Data represent the mean ± standard error of the mean for three different experiments (n = 3). *P ≤ 0.05 vs control, ** P ≤ 0.05 vs lipofection eNOS-siRNA.
Fig 7.
Schematic representation of eNOS-siRNA delivery by UMMD treatment to inhibit angiogenesis in blood vessels.
(A) eNOS is constitutively expressed in endothelial cells. Stimuli such as Vascular Endothelial Growth Factor (VEGF) promote expression and activation of eNOS, inducing NO production. NO promotes endothelial cell migration, proliferation, and tube formation, which are crucial steps in the formation of new blood vessels (angiogenesis). (B) When MBs loaded with eNOS siRNA are directed towards the blood vessel and are stimulated with US, this US causes the rupture of MBs in a process called sonoporation, which facilitates the delivery of eNOS siRNA into the endothelial cells lining the blood vessel. Inside the endothelial cells, the eNOS-siRNA binds to the RNA-induced silencing (RISC) complex, leading to the cleavage and degradation of eNOS mRNA. This downregulates the expression of eNOS, resulting in decreased levels of NO and, consequently, inhibition of angiogenesis. Diagrams were generated in BioRender (https://biorender.com; 2024).