Abstract
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 via 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.