Effects of insonated phospholipids-based microbubbles on the transfection of Antisense Oligonucleotides

摘要

Objective To investigate the effects of insonated phospholipids-based microbubbles on the transfection of Antisense Oligonucleotides. Methods An AS-ODNs sequence HA2741 combined with luciferase reporter plasmid and a breast cancer cell line SK-BR-3 were used. Some important factors on HA2741 transfection efficiency such as wave intensity, ultrasound (US) duration, microbubble concentration and HA2741 concentration, were tested respectively. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. RT-PCR and immunocytochemistry were used to detect the inhibitory effect of HA2741 on HER-2 expression. Atomic force microscopy scanning techniques was employed to observe the change of membrane pore size. Results AS-ODNs transfection efficiency showed an increasing tend with wave intensity, US duration, microbubble concentration and HA2741 concentration, but cell viability showed a negative relationship with these factors. When US with phospholipids-based microbubbles, cell membrane showed many pores with different sizes. With wave intensity, US duration or microbubble concentration increasing, the number of these membrane pores increased and the size of these membrane pores enlarged. Considering all the factors investigated, the maximum transfection efficiency with minimum cell viability achieved under 2% microbubble mixed with 80nmol/L HA2741 for 30s US exposure at -3.0dB wave intensity, which gave an over-all transfection efficiency exceed 90% and cell viability near 90%. Conclusion US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue.