Ekstrand Frida, Davidsson Bencker Sara, Ruhrmann Sabrina, Yang Yupeng, Ling Charlotte, Prinz Christelle N
Division of Solid State Physics, Lund University, 221 00 Lund, Sweden.
NanoLund, Lund University, 221 00 Lund, Sweden.
Nanoscale. 2025 Sep 15. doi: 10.1039/d5nr02352a.
Transfecting clonal beta cells with large DNA plasmids holds significant promise for diabetes research. Existing transfection techniques like lipofection, viral transduction, and bulk electroporation often face limitations such as low efficiency and cytotoxicity. Nanoelectroporation, which utilizes nanopores or nanostraws and the application of mild electrical pulses, offers a gentle and safe alternative capable of delivering mRNAs and small to medium-sized plasmids. Nevertheless, efficiently transfecting cells with large plasmids this approach remains challenging, and further improvements in efficiency are required. Here, we aimed to fill that need and optimized nanoelectroporation substrate properties to increase the transfection efficiency of GFP plasmids in clonal beta cells. We combined flow cytometry, fluorescence microscopy, and phase holographic microscopy to increase nanopore- and nanostraw transfection efficiency in terms of the delivered plasmid quantity. We found that the porosity needs to be high enough to allow the cells to interface enough nanopores, 200 nm nanopore diameter yielded higher transfection efficiency and lower cell death than 300 nm pores, and that the surface chemistry has a great effect on transfection efficiency due to differences in cell adhesion properties. Nanopores and nanostraws were compared and nanostraws were found to yield the highest immediate transfection efficiency. However, cells expressing GFP after 48 h were fewer than indicated immediately after transfection. We investigated the reasons behind this discrepancy in transfection efficiency assessed immediately- and 48 h after nanoelectroporation. Our results suggest that cells transfected with the most plasmids detach from the substrate within 48 h after transfection. This finding confirms that plasmids are cytotoxic, and it stresses the importance of achieving homogeneous transfection efficiencies among cells to be able to tune the amount of delivered plasmids appropriately.
用大型DNA质粒转染克隆β细胞对糖尿病研究具有重大意义。现有的转染技术，如脂质体转染、病毒转导和批量电穿孔，常常面临效率低和细胞毒性等局限性。纳米电穿孔利用纳米孔或纳米吸管并施加温和电脉冲，提供了一种温和且安全的替代方法，能够递送mRNA以及中小型质粒。然而，用这种方法高效转染大型质粒的细胞仍然具有挑战性，需要进一步提高效率。在此，我们旨在满足这一需求，并优化纳米电穿孔底物特性以提高绿色荧光蛋白（GFP）质粒在克隆β细胞中的转染效率。我们结合流式细胞术、荧光显微镜和相全息显微镜，从递送的质粒数量方面提高纳米孔和纳米吸管的转染效率。我们发现孔隙率需要足够高，以使细胞与足够多的纳米孔接触，200纳米直径的纳米孔比300纳米的孔产生更高的转染效率和更低的细胞死亡率，并且由于细胞粘附特性的差异，表面化学对转染效率有很大影响。对纳米孔和纳米吸管进行了比较，发现纳米吸管产生的即时转染效率最高。然而，48小时后表达GFP的细胞比转染后立即显示的要少。我们研究了纳米电穿孔后立即和48小时评估的转染效率差异背后的原因。我们的结果表明，转染质粒最多的细胞在转染后48小时内从底物上脱离。这一发现证实了质粒具有细胞毒性，并强调了在细胞间实现均匀转染效率以能够适当调节递送质粒量的重要性。
