Gene Delivery By Electroporation Using Nanowire Structured Electrodes

In recent years,great efforts have been made in the area of cancer immunotherapy especially the CAR-T technology which has been shown to be successful for the treatment of blood cancers.For CAR-T cell preparation,viral transfection is widely used,but the viral method is complicated and there exists considerable safety risks.Therefore,the development and application of a safe and efficient method for introduction of exogenous nucleic acids into T cells remain to be a challenging,yet critical problem.Compared to viral based transfection methods,physical transfection methods like electroporation is considered as a promising alternative with better safety and wider application area.It may be able to transfect different cell types,including primary cells and those hard to be transfected using viral methods.But currently most electroporation devices use very high voltage(>200V),which may result in serious thermal and electrochemical effects causing poor cell survival after electroporation.In this study,we propose to take advantage of the special electric fields surrounding nanowire structures.It was estimated the electric field in the vicinity may be 2-3 orders of magnitude stronger.We therefore designed a new device to use electrodes with nanowire structure on the surface and tested cell electroporation using very low voltages across the electrodes.We firstly optimized conditions for fabricating CuO nanowires by thermal oxidation.These CuO electrodes were placed in parallel and various electric pulses were applied.Different cells including the CHO cell,RAW264.7,and Jurkat cell lines were treated and plasmid DNA encoding luciferase was used as the reporter gene.The electric pulse parameters varied and their effects were evaluated.Our data demonstrated that plasmid DNA can indeed be able to enter some cells at voltages as low as 2V.Although the transfection efficiency was still low,our work provided the basis for further studies to optimize the design of electrodes and develop protocols for transfecting different cells with minimum perturbation to their normal functions.