Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

Abstract

Aim: To explore the effects of ultrasound exposure combined with microbubble contrast agent (SonoVue) on the permeability of the cellular membrane and on the expression of plasmid DNA encoding enhanced green fluorescent protein (pEGFP) transfer into human umbilical vein endothelial cells (HUVECs).

Methods: HUVECs with fluorescein isothiocyanate-dextran (FD500) and HUVECs with pEGFP were exposed to continuous wave (1.9 MHz, 80.0 mW/cm(2)) for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytometry, respectively.

Results: The percentage of FD500-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone (24.0% +/- 5.5% vs 66.6% +/- 4.1%, P < 0.001). Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue (16.1% +/- 1.9% vs 1.5% +/- 0.2%, P < 0.001). No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue (94.1% +/- 2.3% vs 91.1% +/- 4.1%).

Conclusion: The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non-invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Figure 1

Map of pEGFP-C1, showing the restriction sites.

Figure 2

Experimental setup for the ultrasound exposure. Ultrasound probe with transmission gel was placed on the cell suspension covered with polyester film, a 24-well plate covered by a 6-μm polyester film was kept in a water bath at 37°C, and also a sponge mat was placed in the water bath.

Figure 3

Fluorescent microscopy showing FD500-positive HUVECs (200 ×). (A) A few FD500-positive HUVECs with ultrasound only; (B) significant increase in FD500-positive HUVECs after the addition of SonoVue.

Figure 4

Fluorescent microscopy after EGFP gene transfection (200 ×). (A) EGFP expression 48 h after ultrasound exposure only; (B) significant increase in EGFP expression in HUVECs 48 h after the addition of SonoVue.

Figure 5

Detection of EGFP gene transfection by flow cytometry. (A) Cells exposed to ultrasound only showed 1.5% of HUVECs within M₁ area; (B) cells exposed to both the ultrasound and SonoVue showed 16.1% of HUVECs within M₁ area.

Figure 6

HUVECs survival rate 48 h after gene transfection. ^aP < 0.05 vs group A and B ; ^cP > 0.05 vs group C.