Interrogation on the Cellular Nano-Interface and Biosafety of Repeated Nano-Electroporation by Nanostraw System

Cell perforation is a critical step for intracellular drug delivery and real-time biosensing of intracellular signals. In recent years, the nanostraws system has been developed to achieve intracellular drug delivery with minimal invasiveness to the cells. Repeated cell perforation via nano-system could allow delivery of multiple drugs into cells for cell editing, but the biosafety is rarely explored. In this work, a nanostraw-mediated nano-electroporation system was developed, which allowed repeated perforation of the same set of cells in a minimally invasive manner, while the biosafety aspect of this system was investigated. Highly controllable fabrication of Al(2)O(3) nanostraw arrays based on a porous polyethylene terephthalate (PET) membrane was integrated with a microfluidic device to construct the nanostraw-electroporation system. The pulse conditions and intervals of nano-electroporation were systematically optimized to achieve efficient cells perforation and maintain the viability of the cells. The cells proliferation, the early apoptosis activities after nanostraw-electroporation and the changes of gene functions and gene pathways of cells after repeated nano-electroporation were comprehensively analyzed. These results revealed that the repeated nanostraw-electroporation did not induce obvious negative effects on the cells. This work demonstrates the feasibility of repeated nano-electroporation on cells and provides a promising strategy for future biomedical applications.