Department of Biomedical Engineering, Tianjin University, Tianjin 300072, China.
College of Life Sciences, Nankai University, Tianjin 300071, China.
J Control Release. 2018 Mar 10;273:40-50. doi: 10.1016/j.jconrel.2018.01.014. Epub 2018 Feb 6.
Ultrasound application combined with microbubbles has shown great potential for intracellular gene delivery. However, the fundamental mechanistic question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubble has not been fully explored and understood. The goal of this study is to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, uniquely highlighting the role of microbubbles play in this process. The usage of targeted microbubbles pinpointed the subcellular membrane site, where ultrasound exerted acoustic force onto the cell membrane. With the combination of high-speed video microscopy and 3D confocal fluorescence microscopy, we show the spatiotemporal correlation between the microbubble dynamics and intracellular plasmid DNA distribution. Two ultrasound modes (high pressure short pulse and low pressure long pulse) were chosen to trigger different plasmid DNA uptake routes. We found that reversible cell membrane disruption, induced by high pressure short pulse ultrasound, permitted plasmid DNA passage across cell membrane, but not in an exclusive way. Under both ultrasound modes, with or without cell membrane disruption, global plasmid DNA internalization, even nuclear-localization, was observed immediately post ultrasound application. Our results show that plasmid DNA uptake evoked by localized acoustically excited microbubbles is a fast (<2min), global (not limited to the site where microbubbles were attached), and multi-mechanisms involved process.
超声应用联合微泡在细胞内基因传递方面显示出巨大的潜力。然而，超声和微泡介导的质粒 DNA 如何进入细胞内空间的基本机制问题尚未得到充分探索和理解。本研究旨在揭示超声和微泡刺激下质粒 DNA 的详细细胞内摄取过程，特别强调微泡在这一过程中的作用。靶向微泡的使用定点在超声对细胞膜施加声力的亚细胞膜部位。通过高速视频显微镜和 3D 共聚焦荧光显微镜的结合，我们展示了微泡动力学与细胞内质粒 DNA 分布之间的时空相关性。选择了两种超声模式（高压短脉冲和低压长脉冲）来触发不同的质粒 DNA 摄取途径。我们发现，高压短脉冲超声诱导的可逆细胞膜破裂允许质粒 DNA 通过细胞膜，但不是唯一途径。在两种超声模式下，无论是否破坏细胞膜，都可以观察到质粒 DNA 在超声应用后立即被整体内化，甚至核内定位。我们的结果表明，局部声激发微泡引起的质粒 DNA 摄取是一个快速（<2 分钟）、整体（不限于微泡附着的部位）和多机制参与的过程。
