|
|
|
| Effect of Pulsed Electric Field on Electroporation Area of Cell Membrane |
| Wu Xiao1,2, Mi Yan1, Zheng Wei1, Ma Chi1, Zhang Mengnan1 |
1. State Key Laboratory of Power Transmission Equipment & System Security and New Technology Chongqing University Chongqing 400030 China;
2. Marketing Service Center of State Grid Henan Electric Power Company (Metrology Center) Zhengzhou 450000 China |
|
|
|
Abstract In the field of electrobiology, simulation and experimental methods can be used to study the cell electroporation effect under pulsed electric field. In simulation, parameters such as transmembrane voltage, current density, pore density, pore radius and pore area are usually used to characterize the electroporation effect. In the experiment, the killing effect or the overall situation of the electric field on the cells is usually characterized macroscopically, which is not equivalent to the specific parameters such as the number and size of the pores formed, and it is not easy to establish a close relationship between simulation and experiment. Therefore, the electroporation area of cell membrane, a new characterization parameter to directly measure the electroporation effect, and its experimental measurement method are proposed in this paper.
Firstly, the measurement principle of cell electroporation area was analyzed based on the molecular transport dynamics, and it was found that the electroporation area could be measured and calculated by cell fluorescence experiment. Then, the fluorescent molecular staining experiment was carried out based on the above principle, the fluorescence intensity changes of cells under the electric field were captured in real time under the fluorescence microscope. Before carrying out cell electroporation experiments, the cell fluorescence intensity needs to be corrected to obtain the relationship between the cell fluorescence intensity and the intracellular concentration of propidium iodide ions. Next, the maximum cell fluorescence intensity and the pore recovery time constant were extracted from the fluorescence intensity curve under the action of the electric field, from which the electroporation area of cell membrane formed during the pulse could be calculated. Finally, experiments were carried out under different pulsed electric field parameters (field strength and pulse width) to study the relationship and law between cell electroporation area and pulsed electric field parameters, and the obtained laws were analyzed and explained theoretically.
Under the pulsed electric field parameters selected in this paper, the electroporation area calculated by the cell fluorescent staining experiment is in the order of 10-15 m2, which is quantitatively close to the value calculated in the literature. Under different pulse electric field parameters, with the increase of pulse electric field parameters (field strength and pulse width), the fluorescence intensity of cells increases, the fluorescence intensity curve moves up, and the electroporation effect is enhanced. Moreover, the calculated electroporation area increases with the increase of pulse field strength, pulse width and pulse electric shock, and all have a linear relationship. The obtained law is analyzed in combination with theory, and the laws obtained in this paper are qualitatively consistent with those in the existing literature. By comparing the electroporation area between this paper and the existing conclusions quantitatively and qualitatively, the accuracy of the method and results of calculating the cell electroporation area by experimental measurements can be evaluated. According to these laws, it can provide guidance for the pulsed electric field treatment. For example, in the electrochemotherapy, the pulsed electric field parameters (field strength and pulse width) are increased linearly, the electroporation area is increased, and more drugs enter the cells, and the treatment is more effective.
|
|
Received: 10 May 2022
|
|
|
|
|
|
[1] Kinosita K, Tsong T Y.Formation and resealing of pores of controlled sizes in human erythrocyte membrane[J]. Nature, 1977, 268(5619): 438-441.
[2] Zimmermann U, Pilwat G, Riemann F.Dielectric breakdown of cell membranes[J]. Biophysical Journal, 1974, 14(11): 881-899.
[3] 姚陈果, 宁郡怡, 刘红梅, 等. 微/纳秒脉冲电场靶向不同尺寸肿瘤细胞内外膜电穿孔效应研究[J]. 电工技术学报, 2020, 35(1): 115-124.
Yao Chenguo, Ning Junyi, Liu Hongmei, et al.Study of electroporation effect of different size tumor cells targeted by micro-nanosecond pulsed electric field[J]. Transactions of China Electrotechnical Society, 2020, 35(1): 115-124.
[4] 米彦, 李盼, 刘权, 等. 纳秒脉冲电场联合多壁碳纳米管对皮肤癌细胞活性的剂量效应研究[J]. 电工技术学报, 2019, 34(22): 4849-4857.
Mi Yan, Li Pan, Liu Quan, et al.Dose effect of the activity of skin cancer cells treated by nanosecond pulsed electric field combined with multi-walled carbon nanotubes[J]. Transactions of China Electrotechnical Society, 2019, 34(22): 4849-4857.
[5] Polajžer T, Dermol-Černe J, Reberšek M, et al. Cancellation effect is present in high-frequency reversible and irreversible electroporation[J]. Bioelectrochemistry, 2020, 132: 107442.
[6] Sadik M M, Li Jianbo, Shan J W, et al.Quantification of propidium iodide delivery using millisecond electric pulses: experiments[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2013, 1828(4): 1322-1328.
[7] Dermol J, Miklavčič D.Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination[J]. Bioelectrochemistry, 2014, 100: 52-61.
[8] 米彦, 吴晓, 徐进, 等. 细胞膜力学性能对电穿孔影响的仿真研究[J]. 电工技术学报, 2022, 37(17): 4326-4334, 4400.
Mi Yan, Wu Xiao, Xu Jin, et al.Simulation study on the effect of cell membrane mechanical properties on electroporation[J]. Transactions of China Electrote-chnical Society, 2022, 37(17): 4326-4334, 4400.
[9] 程显, 陈硕, 吕彦鹏, 等. 纳秒脉冲作用下核孔复合体影响细胞核膜电穿孔变化的仿真研究[J]. 电工技术学报, 2021, 36(18): 3821-3828.
Cheng Xian, Chen Shuo, Lü Yanpeng, et al.Simulation study on the effect of nuclear pore complexes on cell electroporation under nanosecond pulse[J]. Transactions of China Electrotechnical Society, 2021, 36(18): 3821-3828.
[10] 胡亚楠, 包家立, 朱金俊, 等. 纳秒电脉冲对肝脏组织不可逆电穿孔消融区分布的时域有限差分法仿真[J]. 电工技术学报, 2021, 36(18): 3841-3850.
Hu Yanan, Bao Jiali, Zhu Jinjun, et al.The finite difference time domain simulation of the distribution of irreversible electroporation ablation area in liver tissue by nanosecond electrical pulse[J]. Transactions of China Electrotechnical Society, 2021, 36(18): 3841-3850.
[11] DeBruin K A, Krassowska W. Modeling electroporation in a single cell. Ⅰ. Effects of field strength and rest potential[J]. Biophysical Journal, 1999, 77(3): 1213-1224.
[12] Liu Hongmei, Yao Chenguo, Mi Yan, et al.Multi-parameter analysis in single-cell electroporation based on the finite element model[C]//2016 IEEE International Power Modulator and High Voltage Conference (IPMHVC), San Francisco, CA, USA, 2016: 364-370.
[13] 刘红梅, 董守龙, 宁郡怡, 等. 纳秒脉冲高频透膜效应优先杀伤化疗抗性肿瘤细胞的仿真与实验研究[J]. 电工技术学报, 2019, 34(22): 4839-4848.
Liu Hongmei, Dong Shoulong, Ning Junyi, et al.Simulation and experimental study on preferential killing of chemoresistance tumor cells induced by the high-frequency permeation effect of nanosecond pulse field[J]. Transactions of China Electrotechnical Society, 2019, 34(22): 4839-4848.
[14] 吕彦鹏. 协同脉冲电场增强不可逆电穿孔组织消融疗效的电学机理与实验研究[D]. 重庆: 重庆大学, 2019.
[15] 米彦, 徐进, 刘宏亮, 等. 基于网格传输网络模型的高频纳秒脉冲串作用下单细胞穿孔特性仿真[J]. 电工技术学报, 2018, 33(18): 4217-4230.
Mi Yan, Xu Jin, Liu Hongliang, et al.Simulation on perforation features in a single-cell system exposed to high-frequency nanosecond pulse bursts based on mesh transport network model[J]. Transactions of China Electrotechnical Society, 2018, 33(18): 4217-4230.
[16] 李成祥, 李新皓, 柯强, 等. 纳/微秒复合脉冲提高细胞电融合效率的仿真与实验研究[J]. 电工技术学报, 2022, 37(6): 1522-1532.
Li Chengxiang, Li Xinhao, Ke Qiang, et al.Simulation and experimental research on improving the efficiency of cell electrofusion by nano-microsecond pulsed electric fields[J]. Transactions of China Electrotechnical Society, 2022, 37(6): 1522-1532.
[17] Batista Napotnik T, Miklavčič D.In vitro electroporation detection methods-an overview[J]. Bioelectrochemistry, 2018, 120: 166-182.
[18] Pucihar G, Kotnik T, Miklavčič D, et al.Kinetics of transmembrane transport of small molecules into electropermeabilized cells[J]. Biophysical Journal, 2008, 95(6): 2837-2848.
[19] Neumann E, Toensing K, Kakorin S, et al.Mechanism of electroporative dye uptake by mouse B cells[J]. Biophysical Journal, 1998, 74(1): 98-108.
[20] Djuzenova C S, Zimmermann U, Frank H, et al.Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1996, 1284(2): 143-152.
[21] Yao Chenguo, Dong Shoulong, Zhao Yajun, et al.High-frequency composite pulse generator based on full-bridge inverter and soft switching for biological applications[J]. IEEE Transactions on Dielectrics and Electrical Insulation, 2016, 23(5): 2730-2737.
[22] Kennedy S M, Ji Z, Hedstrom J C, et al.Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells[J]. Biophysical Journal, 2008, 94(12): 5018-5027.
[23] Sukhorukov V L, Djuzenova C S, Frank H, et al.Electropermeabilization and fluorescent tracer exchange: the role of whole-cell capacitance[J]. Cytometry, 1995, 21(3): 230-240.
[24] Schoenbach K H, Joshi R P, Beebe S J, et al.A scaling law for membrane permeabilization with nanopulses[J]. IEEE Transactions on Dielectrics and Electrical Insulation, 2009, 16(5): 1224-1235.
[25] Dermol-Černe J, Miklavčič D, Reberšek M, et al. Plasma membrane depolarization and permeabilization due to electric pulses in cell lines of different excitability[J]. Bioelectrochemistry, 2018, 122: 103-114.
[26] Geboers B, Scheffer H J, Graybill P M, et al.High-voltage electrical pulses in oncology: irreversible electroporation, electrochemotherapy, gene electrotransfer, electrofusion, and electroimmunotherapy[J]. Radiology, 2020, 295(2): 254-272.
[27] Liu Hongmei, Yao Chenguo, Zhao Yajun, et al.In vitro experimental and numerical studies on the preferential ablation of chemo-resistant tumor cells induced by high-voltage nanosecond pulsed electric fields[J]. IEEE Transactions on Bio-Medical Engineering, 2021, 68(8): 2400-2411.
[28] Glaser R W, Leikin S L, Chernomordik L V, et al.Reversible electrical breakdown of lipid bilayers: formation and evolution of pores[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1988, 940(2): 275-287.
[29] Taupin C, Dvolaitzky M, Sauterey C.Osmotic pressure induced pores in phospholipid vesicles[J]. Biochemistry, 1975, 14(21): 4771-4775.
[30] Kotnik T, Rems L, Tarek M, et al.Membrane electroporation and electropermeabilization: mechanisms and models[J]. Annual Review of Biophysics, 2019, 48: 63-91.
[31] Chen C, Smye S W, Robinson M P, et al.Membrane electroporation theories: a review[J]. Medical and Biological Engineering and Computing, 2006, 44(1): 5-14.
[32] Muralidharan A, Rems L, Kreutzer M T, et al.Actin networks regulate the cell membrane permeability during electroporation[J]. Biochimica et Biophysica Acta Biomembranes, 2021, 1863(1): 183468.
[33] Kinosita K, Ashikawa I, Saita N, et al.Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope[J]. Biophysical Journal, 1988, 53(6): 1015-1019.
[34] Dimova R, Riske K A, Aranda S, et al.Giant vesicles in electric fields[J]. Soft Matter, 2007, 3(7): 817-827.
[35] Ahamed M K, Abu Sayem Karal M, Ahmed M, et al. Kinetics of irreversible pore formation under constant electrical tension in giant unilamellar vesicles[J]. European Biophysics Journal, 2020, 49(5): 371-381.
[36] 贡凌宇. 基于微流控芯片的单细胞电穿孔仿真与实验研究[D]. 重庆: 重庆大学, 2017. |
|
|
|
2023, Vol. 38 
