Mowla Mahshid, Gorji-Bahri Gilar, Moghimi Hamid Reza, Hashemi Atieh
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.
Section of Medical Protein Chemistry, Department of Translational Medicine, Lund University, Malmö, Sweden.
Res Pharm Sci. 2024 Dec 15;19(6):766-773. doi: 10.4103/RPS.RPS_185_23. eCollection 2024 Dec.
Intracellular delivery is crucial in biological and medical studies. Although many molecular tools have been created for cell-based gene therapies, it remains challenging to introduce external molecules into cells. As one of the most popular non-viral transfection methods, electroporation induces transient pores in the cell membrane by applying an external electric field. Unsatisfactory transfection efficiency and low cell viability are the major drawbacks of electroporation. To overcome these issues, the current study investigated the effect of urea on electroporation-mediated transfection efficiency.
Three voltages of electroporation, including 100, 120, and 140 V, and 3 concentrations of urea buffer, including 0.25%, 0.5%, and 1% W/V, were considered as variables in this study. The HEK-293 cell line was used for transfection, and green fluorescent protein (GFP) expression was evaluated using flow cytometry and fluorescence microscopy.
FINDINGS/RESULTS: The results showed that the combination of electroporation and urea increased electroporation efficacy, but the effect depended on voltage and urea concentration. When different concentrations of urea were added to HEK-293 cells at a voltage of 100 V, the number of cells transfected by pEGFP-N1 increased (from 12.3 ± 0.2% in untreated cells to 17.35 ± 0.55%, 23.3 ± 0.3%, and 14 ± 0.1% at urea concentrations of 0.25%, 0.5%, and 1% W/V, respectively). The electroporation buffer containing 0.5% W/V urea showed the highest EGFP expression (23.3 ± 0.3%) and high cell viability (over 90%).
This research offers a new perspective for improving gene transfection efficiency once electroporation is utilized.
细胞内递送在生物学和医学研究中至关重要。尽管已经开发了许多用于基于细胞的基因治疗的分子工具，但将外部分子导入细胞仍然具有挑战性。作为最流行的非病毒转染方法之一，电穿孔通过施加外部电场在细胞膜上诱导瞬时孔道。转染效率不理想和细胞活力低是电穿孔的主要缺点。为了克服这些问题，本研究调查了尿素对电穿孔介导的转染效率的影响。
本研究将三种电穿孔电压（100、120和140V）和三种尿素缓冲液浓度（0.25%、0.5%和1%W/V）视为变量。使用HEK-293细胞系进行转染，并通过流式细胞术和荧光显微镜评估绿色荧光蛋白（GFP）的表达。
结果表明，电穿孔与尿素的组合提高了电穿孔效率，但效果取决于电压和尿素浓度。当在100V电压下向HEK-293细胞中添加不同浓度的尿素时，pEGFP-N1转染的细胞数量增加（未处理细胞中为12.3±0.2%，在尿素浓度为0.25%、0.5%和1%W/V时分别为17.35±0.55%、23.3±0.3%和14±0.1%）。含有0.5%W/V尿素的电穿孔缓冲液显示出最高的EGFP表达（23.3±0.3%）和高细胞活力（超过90%）。
本研究为利用电穿孔提高基因转染效率提供了新的视角。
