Ultrasound triggered image-guided drug delivery to inhibit vascular reconstruction via paclitaxel-loaded microbubbles

Abstract

Paclitaxel (PTX) has been recognized as a promising drug for intervention of vascular reconstructions. However, it is still difficult to achieve local drug delivery in a spatio-temporally controllable manner under real-time image guidance. Here, we introduce an ultrasound (US) triggered image-guided drug delivery approach to inhibit vascular reconstruction via paclitaxel (PTX)-loaded microbubbles (PLM) in a rabbit iliac balloon injury model. PLM was prepared through encapsulating PTX in the shell of lipid microbubbles via film hydration and mechanical vibration technique. Our results showed PLM could effectively deliver PTX when exposed to US irradiation and result in significantly lower viability of vascular smooth muscle cells. Ultrasonographic examinations revealed the US signals from PLM in the iliac artery were greatly increased after intravenous administration of PLM, making it possible to identify the restenosis regions of iliac artery. The in vivo anti-restenosis experiments with PLM and US greatly inhibited neointimal hyperplasia at the injured site, showing an increased lumen area and reduced the ratio of intima area and the media area (I/M ratio). No obvious functional damages to liver and kidney were observed for those animals. Our study provided a promising approach to realize US triggered image-guided PTX delivery for therapeutic applications against iliac restenosis.

Figure 1. Characterization of PLM.

(A) Schematic diagram of a PLM constructed for drug delivery. PTX was encapsulated into the shell of gas-filled MBs. (B) A typical bottle of PLM after mechanical activation. (C) An optical microscope of PLM, scale bar = 10 μm. (D) The bubble size distributions of the control MBs and PLM. (E–G) The MB concentrations, bubble size and drug EE of PLM after the freshly prepared PLMs were left to stand for 0.5, 6, 18 or 32 h.

Figure 2. Drug release and in vitro cell growth inhibition.

(A) US promoted PTX release from PLM in vitro. *P < 0.05. (B) Cell viability of VSMC after treatment with PBS, US, LM + US, PLM or PLM + US. After different interventions, the cells were cultured for another 24 h at 37 °C. Cell viability was determined by CCK-8 assay. *P < 0.05,**P < 0.01.

Figure 3. US imaging of PLM and Sonovue in the rabbit iliac arteries.

(A) The rabbits were administrated with PLM (Top two rows) or Sonovue (bottom two rows) at the same bubble concentrations. After bubble perfusion, US images were obtained at 0.1 s, 0.5 s, 1 s, 3 s or 10 s. (B,C) The quantitative analysis of US signal intensities of selected regions after PLM or Sonovue perfusion at different times. The arrows are for the regions of iliac artery restenosis. Red boxes are for selected regions of the rabbit iliac arteries. White boxes for selected regions of tissue background. *P < 0.01.

Figure 4. Overview of the experimental protocol.

(A) Experimental protocol over time (days) is shown, with each vertical line on the x-axis representing a time point at intervals of one week. The arrows represent the time point of animal treatments, corresponding to balloon injury, start therapy and evaluation, respectively. (B) Diagram shows the experimental setting, with the rabbit lying supine and the site of iliac artery restenosis received with US imaging and therapy. Partial enlarged drawing of iliac artery restenosis which was perfused with PLM and received with therapy under US imaging is shown (inset). (C) The isolated fragments of the iliac artery with restenosis and without restenosis were presented. Scale bar: 2 mm. (D) The in vivo drug local delivery efficiency was determined by examining PTX amounts in the isolated iliac arteries.

Figure 5. Efficacy assessment of iliac artery restenosis.

(A) Representative H&E-stained arterial cross-sections from different treatment groups in a rabbit model of iliac artery restenosis (top). Zoomed-in H&E-stained arterial cross-sections highlight the different vascular remodeling from the different treatment groups (bottom). I, intima; M, media; E, externa. Scale bar, 100 μm. (B,C) Quantitative analysis of luminal area and intima-to-media area ratio from the different treatment groups. (D,E) Representative zoomed-in H&E-stained arterial cross-sections from PLM + US group and control group highlight the significant reduction of disorder cell arrangement and irregular cell shape (red arrows). Scale bar: 100 μm.

Figure 6. Immunohistochemical staining for α-SMA proteins.

(A) Representative anti-α-SMA antibody-stained arterial cross-sections from different treatment groups in a rabbit model of iliac artery restenosis. Scale bar, 50 μm. (B,C) Quantitative analysis of α-SMA-positive cells and the immunohistochemical score (HIS) from the different treatment groups. Representative zoomed-in H&E-stained arterial cross-sections from PLM + US group (D) and control group (E) highlight the significant reduction of intimal hyperplasia and irregularly shaped cells (red arrows). Scale bar: 100 μm.