Shamout Farah E, Pouliopoulos Antonios N, Lee Patrizia, Bonaccorsi Simone, Towhidi Leila, Krams Rob, Choi James J
Department of Bioengineering, Imperial College London, London, UK.
Department of Bioengineering, Imperial College London, London, UK.
Ultrasound Med Biol. 2015 Sep;41(9):2435-48. doi: 10.1016/j.ultrasmedbio.2015.05.003. Epub 2015 Jun 9.
Sonoporation has been associated with drug delivery across cell membranes and into target cells, yet several limitations have prohibited further advancement of this technology. Higher delivery rates were associated with increased cellular death, thus implying a safety-efficacy trade-off. Meanwhile, there has been no reported study of safe in vitro sonoporation in a physiologically relevant flow environment. The objective of our study was not only to evaluate sonoporation under physiologically relevant flow conditions, such as fluid velocity, shear stress and temperature, but also to design ultrasound parameters that exploit the presence of flow to maximize sonoporation efficacy while minimizing or avoiding cellular damage. Human umbilical vein endothelial cells (EA.hy926) were seeded in flow chambers as a monolayer to mimic the endothelium. A peristaltic pump maintained a constant fluid velocity of 12.5 cm/s. A focused 0.5 MHz transducer was used to sonicate the cells, while an inserted focused 7.5 MHz passive cavitation detector monitored microbubble-seeded cavitation emissions. Under these conditions, propidium iodide, which is normally impermeable to the cell membrane, was traced to determine whether it could enter cells after sonication. Meanwhile, calcein-AM was used as a cell viability marker. A range of focused ultrasound parameters was explored, with several unique bioeffects observed: cell detachment, preservation of cell viability with no membrane penetration, cell death and preservation of cell viability with sonoporation. The parameters were then modified further to produce safe sonoporation with minimal cell death. To increase the number of favourable cavitation events, we lowered the ultrasound exposure pressure to 40 kPapk-neg and increased the number of cavitation nuclei by 50 times to produce a trans-membrane delivery rate of 62.6% ± 4.3% with a cell viability of 95% ± 4.2%. Furthermore, acoustic cavitation analysis showed that the low pressure sonication produced stable and non-inertial cavitation throughout the pulse sequence. To our knowledge, this is the first study to demonstrate a high drug delivery rate coupled with high cell viability in a physiologically relevant in vitro flow system.
声孔效应已与跨细胞膜并进入靶细胞的药物递送相关联，但一些限制因素阻碍了该技术的进一步发展。较高的递送率与细胞死亡增加相关，这意味着存在安全性与有效性之间的权衡。与此同时，尚未有关于在生理相关流动环境中进行安全的体外声孔效应的研究报道。我们研究的目的不仅是评估在生理相关流动条件下（如流体速度、剪切应力和温度）的声孔效应，还在于设计超声参数，利用流动的存在来最大化声孔效应效率，同时最小化或避免细胞损伤。将人脐静脉内皮细胞（EA.hy926）以单层形式接种在流动腔室中以模拟内皮。蠕动泵维持12.5厘米/秒的恒定流体速度。使用聚焦的0.5兆赫兹换能器对细胞进行超声处理，同时插入的聚焦7.5兆赫兹被动空化探测器监测接种微泡后的空化发射。在这些条件下，追踪通常不能透过细胞膜的碘化丙啶，以确定超声处理后它是否能进入细胞。与此同时，使用钙黄绿素 - AM作为细胞活力标记物。探索了一系列聚焦超声参数，观察到了几种独特的生物效应：细胞脱离、在无膜穿透情况下保持细胞活力、细胞死亡以及通过声孔效应保持细胞活力。然后进一步修改参数以产生具有最小细胞死亡的安全声孔效应。为了增加有利的空化事件数量，我们将超声暴露压力降低至40 kPapk - neg，并将空化核数量增加50倍，以产生62.6% ± 4.3%的跨膜递送率，细胞活力为95% ± 4.2%。此外，声空化分析表明，低压超声处理在整个脉冲序列中产生稳定且非惯性的空化。据我们所知，这是第一项在生理相关的体外流动系统中证明高药物递送率与高细胞活力相结合的研究。
