[Optimizing the operating variables that affect the transfection experiment of antisense oligodeoxyribonucleotide by gas-filled microbubbles].
To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
Zhao
Ying-zheng
YZ
Department of Clinical Pharmacology, General Hospital of Beijing Military Command, Beijing 100700, China. lctuua@yahoo.com.cn
Luo
Yu-kun
YK
Lu
Cui-tao
CT
Xu
Jing-feng
JF
Mei
Xing-guo
XG
Wang
Hu-jun
HJ
Zhang
Mei
M
D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins; D006801:Humans; D045423:Microbubbles; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D013459:Sulfur Hexafluoride; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
D020319:Oligodeoxyribonucleotides, Antisense; D049452:Green Fluorescent Proteins; D013459:Sulfur Hexafluoride