Enhanced downregulation of transforming growth factor‑β2 in rat retinal pigment epithelium cells by adeno‑associated virus‑mediated ribonucleic acid interference combined with ultrasound or microbubbles.

Abstract

The present study was designed to determine the efficiency and safety of ultrasound (US) and/or US contrast agent microbubbles (MBs) in the delivery of type 2 recombinant adeno-associated virus‑delivered transforming growth factor‑β2 short hairpin ribonucleic acid encoding the enhanced green fluorescent protein gene (rAAV2‑TGFβ2 shRNA‑EGFP) and the downregulation of TGFβ2 in rat retinal pigment epithelium (RPE‑J) cells. The effects of US and/or MBs on the delivery of rAAV2‑EGFP and rAAV2‑TGFβ2 shRNA‑EGFP were evaluated by fluorescence microscopy and flow cytometry. The potential toxicity of cell viability under various US or MB conditions was assessed by CellTiter 96® AQueous One solution cell proliferation assay. The level of TGFβ2 mRNA in RPE‑J cells under various conditions was estimated by reverse transcription‑quantitative polymerase chain reaction analysis. The results obtained demonstrated that low-intensity US (0.5 W/cm2 and 30 sec) or SonoVue (MB:cell ratio, 40:1) increased the delivery efficiency of rAAV2‑EGFP and rAAV2‑TGFβ2 shRNA‑EGFP to RPE‑J cells, whereas the combination of US with MBs did not further increase but instead decreased rAAV transfection. Under the optimal conditions of rAAV delivery, enhanced TGFβ2 gene silencing with a combination of US or SonoVue with rAAV2‑TGFβ2 shRNA resulted in a significant decrease in mRNA levels compared with rAAV2‑TGFβ2 shRNA alone. US or SonoVue was used safely to enhance the delivery of rAAV2‑TGFβ2 shRNA to RPE‑J cells. A combination of the biological (rAAV2‑TGFβ2 shRNA) and physical (US or SonoVue) approaches downregulated the mRNA level of TGFβ2 more effectively.