Department of Mechanical and Aerospace Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey.
Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey.
Biophys J. 2014 Feb 18;106(4):801-12. doi: 10.1016/j.bpj.2013.12.045.
The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.
电穿孔的效果在广泛的生物研究和临床应用中变化显著，但截至目前，文献中尚未提供一种可同时提高效率和保持细胞活力的通用方法。为了解决这一差异，我们在这里概述了一种方法，该方法基于电穿孔介导的分子传递、细胞活力和电脉冲参数之间的比例关系的映射。将荧光素右旋糖苷递送到 3T3 小鼠成纤维细胞中作为模型系统。脉冲被合理地分为两个连续的阶段：第一个是用于渗透的前导脉冲，然后是第二个用于分子传递的脉冲。在第二个脉冲强度和持续时间的参数空间中收集并分析了大量数据，并通过流式细胞术进行了分析。荧光强度与第二个脉冲持续时间呈线性相关，证实电泳在传递中起主导作用。传递效率与场强呈特征性的 S 形依赖性。使用 7-氨基放线菌素 D 对短期细胞死亡进行检查，结果表明与电能之间存在令人信服的线性关系。基于这些比例关系，可以确定最佳场强。还进行了模型研究，并将结果与实验数据进行了比较，以阐明潜在的机制。比较表明，存在一个临界跨膜电位，超过该电位后，第二个脉冲的传递就会有效。这些努力共同建立了一种增强电穿孔功能的通用方法。
