Physical parameters influencing optimization of ultrasound-mediated DNA transfection
Physical parameters influencing optimization of ultrasound-mediated DNA transfection
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DOI：
10.1016/j.ultrasmedbio.2004.01.008
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发表时间：
2004-04-01
影响因子：
2.9
通讯作者：
Prausnitz, MR
中科院分区：
文献类型：
--
作者：
Zarnitsyn, VG;Prausnitz, MR
超声波（US）已被证明可以瞬时破坏细胞膜，从而促进药物和基因加载到活细胞中。为了解决基因治疗应用的优化，本工作的目的是系统地确定两种不同的DNA质粒（pEGFP-N1和pGL 3）的DU 145前列腺癌细胞的转染和活力的物理参数的影响。通过在裸DNA存在下体外超声处理细胞，我们发现转染效率增加：1。在10至30 J/cm（2）下优化声能（对于我们的装置，在高于空化阈值的压力下）; 2.在存在Optison（R）的情况下使用500-kHz US使空化成核，而不是在没有Optison（R）的情况下使用24-kHz US; 3.将细胞浓度从10（6）增加至10（7）个细胞/mL;以及4.在超声处理期间将温度从21 ℃改变到37 ℃。本研究中的最佳条件在没有显著DNA损伤的情况下使转染增加了近100倍。额外的测量表明，少于四分之一的细胞与DNA质粒摄取到胞质溶胶显示DNA表达，这表明进一步优化转染的US可能需要促进细胞内DNA运输。（电子邮件：马克。chbe.gatech.edu）（C）2004年世界医学生物学超声联合会。
Ultrasound (US) has been shown to transiently disrupt cell membranes and, thereby, facilitate the loading of drugs and genes into viable cells. To address optimization of gene therapy applications, the aim of this work was to systematically determine the influence of physical parameters on transfection and viability of DU145 prostate cancer cells by two different DNA plasmids (pEGFP-N1 and pGL3). By sonicating cells in vitro in the presence of naked DNA, we found that transfection efficiency was increased by: 1. optimizing acoustic energy at 10 to 30 J/cm(2) (for our apparatus, at pressures above the cavitation threshold); 2. using 500-kHz US in the presence of Optison(R) to nucleate cavition, rather than 24-kHz US without Optison(R); 3. increasing cell concentration from 10(6) to 10(7) cells/mL; and 4. changing temperature during sonication from 21 to 37degreesC. The best conditions in this study increased transfection by almost 100-fold in the absence of significant DNA damage. Additional measurements indicated that less than one fourth of cells with DNA plasmid uptake into the cytosol showed DNA expression, which suggests that further optimizing transfection by US may require facilitating intracellular DNA trafficking. (E-mail: mark. prausnitz @ chbe.gatech.edu) (C) 2004 World Federation for Ultrasound in Medicine Biology.