Department of Biophysics, Federal University of São Paulo, Brazil.
J Gene Med. 2011 Jul;13(7-8):392-401. doi: 10.1002/jgm.1586.
Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process.
NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7 kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay.
US at 2 W/cm(2) with a duty-cycle of 20% for 30 s resulted in approximately 40% transfection efficiency but, at 1 W/cm(2) , resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1 W/cm(2) . US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1.
The present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis.
超声（US）已广泛用于提高非病毒载体转染的效率。质粒摄取的机制通常归因于声孔作用，尽管没有明确的证据支持这种归因。基于我们之前的结果，我们假设其他机制，如内吞作用，可能参与这一过程。
使用无微泡的治疗性超声，将质粒载体 pEGFP-N3（4.7kb）转染 NIH3T3 细胞。在用超声照射后，通过共聚焦显微镜实时用荧光染料检测生物效应，如钙内流、活性氧（ROS）的产生和膜电位的改变。使用免疫荧光测定法，还通过内吞作用标记物监测细胞内标记质粒 DNA 的定位。
2W/cm(2)的超声强度，20%的占空比，持续 30s，可使转染效率达到约 40%，而 1W/cm(2)的超声强度则导致非常低的转染水平。尽管在关闭超声后不久，ROS 的产生和钙内流都停止了，但在关闭超声后不久，ROS 的产生和钙内流都增加了，除了 1W/cm(2)的钙内流。超声还将细胞膜电位变为超极化状态，在超声照射后很快恢复到正常状态。标记的质粒 DNA 可以与网格蛋白介导的内吞作用标记物共定位，但不能与 caveolin-1 共定位。
本研究数据表明，超声促进的质粒 DNA 摄取应通过网格蛋白介导的内吞作用发生。
