Zaharoff David A, Henshaw Joshua W, Mossop Brian, Yuan Fan
Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Durham, North Carolina 27708, USA.
Exp Biol Med (Maywood). 2008 Jan;233(1):94-105. doi: 10.3181/0704-RM-113.
Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.
脉冲电场已被广泛用作非病毒基因递送平台。通过对质粒DNA的孔动力学和细胞内转运进行定量分析，可以提高递送效率。为此，我们研究了电穿孔过程中大分子的细胞摄取机制。在该研究中，将分子量为4000（FD-4）或2000000（FD-2000）的异硫氰酸荧光素标记的葡聚糖（FD）在施加不同脉冲电场之前或在不同时间点（即1、2或10秒）添加到小鼠乳腺癌细胞（4T1）的悬浮液中（在高压模式下：振幅为1.2-2.0 kV，持续时间为99微秒，脉冲数为1-5个；在低压模式下：振幅为100-300 V，持续时间为5-20毫秒，脉冲数为1-5个）。使用共聚焦显微镜技术对FD的细胞内浓度进行定量。为了理解转运机制，开发了一个数学模型用于细胞摄取的数值模拟。我们观察到，FD-2000的最大细胞内浓度低于脉冲介质中浓度的3%。细胞内浓度随脉冲数和振幅呈线性增加。此外，在相同的脉冲条件下，FD-2000的细胞内浓度比FD-4低约40%。数值模拟预测，如果模拟浓度与实验数据处于同一数量级，那么在施加脉冲电场后，大于FD-4的孔持续时间<10毫秒。此外，模拟结果表明，对于FD分子的细胞摄取，扩散可忽略不计。综上所述，数据表明脉冲电场在膜上诱导产生的大孔在脉冲施加后迅速消失，对流可能是不带电大分子细胞摄取的主要转运方式。
