Core to the prospects of cellular engineering for disease treatment are facile, yet tunable, methods to load cells with a range of therapeutic cargo. The process ideally allows for delivery to many cells at once, and it maintains cargo and cell faculties after transport. Nanopore electroporation (NanoEP) offers a gentler, scalable, and more precise alternative for cell cargo manipulations. It utilizes an electrically insulating membrane (PCTE) with cylindrical, nanoscale pores whose size, shape, and distribution determine the membrane’s local electric field. This membrane sits on the electrode and localizes the applied voltage to permeate phospholipid bilayer membranes of cells growing on it. This process technology improves viability and delivery outcomes with a lower driving force (15-30 V DC pulses); however, reported efficacy and experimental parameters (i.e. cell density) in literature are system dependent, sometimes with theoretical inconsistencies. These systems typically have variance due to the nature of measuring a complex and indirect biological response, which limits reproducibility and adaptability. One component rarely discussed is how electrode material and electrochemical interactions affect the voltage distribution. We surmise that by taking better account of electrochemical interactions we can better control NanoEP and improve reproducibility. Electrodes can facilitate chemical reactions that negatively affect cell health or hinder cargo manipulation (i.e. pH changes, bubbles). Further, Indium tin oxide (ITO) electrodes are commonly used to maintain visibility, but they have complex electrochemical surfaces, which can influence the local voltage distribution. This dynamic electrochemical environment, combined with uncontrolled cell growth on the membrane, creates unique impedance distributions for every trial and presents a challenge for reproducible process control.
In this work, we investigate the effect of previously overlooked electrode interfacial reactions on NanoEP chemical flux, through a combination of first principles modelling and high-dimensional, experimental data analysis. I utilize electrochemical impedance spectroscopy, scanning electrochemical microscopy (SECM), and in-situ fluorescent visualization to characterize the cellular response to NanoEP and establish correlations between the spatial uniformity of the manipulation to the electrochemical environment. By using optical and electrochemical reporters of the local electric field in our system, we corroborate simulated electric fields and fluxes with experimental results. We find that by lowering surface resistance (i.e. charge transfer resistance), with potassium ferro/ferricyanide as a mediator, we can consistently maintain high flux from cells (>60% cargo removal efficiency), with >90% viability and a 10 fold decrease in the time required, compared to literature. We show that spatial uniformity is more strongly correlated to global geometry and driving force magnitude than electrochemical effects on the voltage distribution.
