Insight concerning the mechanism of therapeutic ultrasound facilitating gene delivery: increasing cell membrane permeability or interfering with intracellular pathways?

Abstract

Nonviral gene delivery methods encounter major barriers in plasmid DNA (pDNA) trafficking toward the nucleus. The present study aims to understand the role and contribution of therapeutic ultrasound (TUS), if any, in pDNA trafficking in primary cells such as fibroblasts and cell lines (e.g., baby hamster kidney [BHK]) during the transfection process. Using compounds that alter the endocytic pathways and the cytoskeletal network, we show that after TUS application, pDNA trafficking in the cytoplasm is not mediated by endocytosis or by the cytoskeletal network. Transfection studies and confocal analyses showed that the actin fibers impeded TUS-mediated transfection in BHK cells, but not in fibroblasts. Flow cytometric analyses indicated that pDNA uptake by cells occurs primarily when the pDNA is added before and not after TUS application. Taken together, these results suggest that TUS by itself operates as a mechanical force driving the pDNA through the cell membrane, traversing the cytoplasmic network and into the nucleus.

FIG. 1.

Effect of endocytic drugs on transfection using therapeutic ultrasound (TUS) and jetPEI. Baby hamster kidney (BHK) cells and fibroblasts were transfected by TUS (30% duty cycle [DC], 2 W/cm², 30 min) and by jetPEI with pLuc, without any inhibitor (control) or with two concentrations of (A) ammonium chloride (AC) or (B) wortmannin (Wort). Results are presented as fold increase in luciferase activity compared with the control group. Cell viability was measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT). n=16; *p<0.05.

FIG. 2.

Localization of DNA in BHK cells or fibroblasts relative to endosomes or lysosomes after TUS or jetPEI transfection. BHK cells (A and B) and fibroblasts (C and D) were transfected by TUS (30% DC, 2 W/cm², 30 min) or jetPEI with fluorescently labeled plasmid, and fixed immediately after TUS or 2 and 5 hr after TUS or jetPEI. Cells without treatment served as controls. (A and C) Endosomes stained with FITC-conjugated anti-EEA1 (green) and pDNA stained with rhodamine (red). (B and D) Lysosomes stained with rhodamine–LysoTracker (red) and pDNA stained with fluorescein (green). Images are representatives of 10 micrographs based on confocal analyses. Scale bars: For BHK cells, 10 μm; for fibroblasts, 20 μm. (E and F) Quantification of colocalization coefficient of pDNA with endosomes or lysosomes in BHK cells (E) and fibroblasts (F). n=10; *p<0.05.

FIG. 3.

Effect of cytoskeletal depolymerization factors on transfection by TUS. (A) BHK cells and (B) fibroblasts were transfected with pLuc, using TUS at 30% DC, 2 W/cm² for 30 min, with and without the addition of cytochalasin B (CytoB) and nocodazole (Noc). Luciferase activity is presented as relative light units (RLU) per milligram of protein and as fold increase compared with control cells (cells treated with TUS plus pLuc without any factor). n=12; *p<0.05.

FIG. 4.

Localization of pDNA in relation to the cytoskeletal network after TUS. TUS was applied at 30% DC, 2 W/cm² for 30 min. (A and B) Effect of CytoB on DNA (red) and actin (green) in (A) BHK cells and (B) fibroblasts. (C and D) Effect of Noc on DNA (green) and microtubules (red) in (C) BHK cells and (D) fibroblasts. Scale bars: For BHK cells, 10 μm; for fibroblasts, 20 μm. Images are representative micrographs of confocal images.

FIG. 4.

Localization of pDNA in relation to the cytoskeletal network after TUS. TUS was applied at 30% DC, 2 W/cm² for 30 min. (A and B) Effect of CytoB on DNA (red) and actin (green) in (A) BHK cells and (B) fibroblasts. (C and D) Effect of Noc on DNA (green) and microtubules (red) in (C) BHK cells and (D) fibroblasts. Scale bars: For BHK cells, 10 μm; for fibroblasts, 20 μm. Images are representative micrographs of confocal images.

FIG. 5.

Effect of DNA addition after TUS on transfection and pDNA uptake by cells. (A) TUS was applied to BHK cells at 30% DC, 2 W/cm² for 30 min, and pLuc was added before TUS or at various times after TUS application. Luciferase activity was measured for 3 days after TUS. n=12. (B) BHK cells were exposed to TUS at 30% DC, 2 W/cm² for 30 min. Rhodamine-labeled plasmid DNA (rpDNA) was added to the cells before TUS, immediately after TUS, or 5 hr after TUS. Cells were incubated for another 24 hr, calcein acetoxymethyl ester (CAM) was added to the cells, and cells were analyzed by fluorescence-activated cell sorting for their relative fluorescence and percentage of pDNA uptake by cells. BHK cells without any treatment, and BHK cells to which CAM and rpDNA were added without TUS, served as controls. **p<0.001.