Single-cell electroendocytosis on a micro chip using in situ fluorescence microscopy

Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent intensity from a large number (> 2,000) of single cells. The EED efficiency was determined as a function of three electric parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the time-dependent mechanism of EED at the single-cell level.