Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries

Abstract

This study tested the hypothesis that ultrasound can target intracellular uptake of drugs into vascular endothelial cells (ECs) at low to intermediate energy and into smooth muscle cells (SMCs) at high energy. Ultrasound-enhanced delivery has been shown to enhance and target intracellular drug and gene delivery in the vasculature to treat cardiovascular disease, but quantitative studies of the delivery process are lacking. Viable ex vivo porcine carotid arteries were placed in a solution containing a model drug, TO-PRO(R)-1, and Optison microbubbles. Arteries were exposed to ultrasound at 1.1 MHz and acoustic energies of 5.0, 66, or 630 J/cm(2). Using confocal microscopy and fluorescent labeling of cells, the artery endothelium and media were imaged to determine the localization and to quantify intracellular uptake and cell death. At low to intermediate ultrasound energy, ultrasound was shown to target intracellular delivery into viable cells that represented 9-24% of exposed ECs. These conditions also typically caused 7-25% EC death. At high energy, intracellular delivery was targeted to SMCs, which was associated with denuding or death of proximal ECs. This work represents the first known in-depth study to evaluate intracellular uptake into cells in tissue. We conclude that significant intracellular uptake of molecules can be targeted into ECs and SMCs by ultrasound-enhanced delivery suggesting possible applications for treatment of cardiovascular diseases and dysfunctions.

Figure 1

(A) Equipment schematic (B) Artery segments were sutured to TPX^® plastic disks exposing the endothelium for flat artery experiments. Confocal images for quantification of bioeffects were captured in a 5×4 array, where each image was spaced by 1.5 mm.

Figure 2

Confocal microscopy 2×2 image montages (10X magnification) displaying the localization of intracellular uptake enhanced by ultrasound. Images depict the endothelium of (A) a control sample and (B) a sample exposed to intermediate ultrasound energy. EC nuclei were labeled with Hoechst 33342 (blue) to stain all cells, propidium iodide (red) to stain dead cells, and TO-PRO^®-1 (green) to indicate intracellular uptake. (A2) and (B2) are shown without blue fluorescence to more clearly view intracellular uptake and cell death.

Figure 3

Confocal microscopy images of the artery surface at 10X (A, C, E, G) and 40X magnification (B, D, G, H) showing the range of bioeffects at different ultrasound energies. Images depict (A–B) a control sample and samples exposed to ultrasound at (C–D) low, (E–F) intermediate, and (G–H) high energies. Figures A2–H2 are shown without blue fluorescence to more clearly view intracellular uptake and cell death.

Figure 4

Quantification of endothelial bioeffects following ultrasound exposure. Data represent the averages of n ≥ 5 replicates with SEM shown.

Figure 5

Confocal microscopy images at multiple depths in the artery displaying bioeffects to medial SMCs. Images were captured at (A) the artery surface and (B) 13 μm and (C) 21 μm below the artery surface.

Figure 6

Confocal microscopy images of endothelium displaying bioeffects mediated by ultrasound exposure to intact arteries at near physiologic conditions. Ultrasound was applied at (A, C) intermediate and (B, D) high energy while the artery was filled with (A, B) DMEM or (C, D) blood.