Limited Efficacy of Nanoparticle-Assisted Electroporation for Membrane Permeabilization and Gene Electrotransfer

<b>Background/Objectives</b>: Nanoparticles (NPs) were previously explored as enhancers in electroporation due to their potential to locally amplify electric fields near cell membranes, with gold nanoparticles (AuNPs) in particular showing promise in improving membrane permeability and gene electrotransfer (GET). In this study, we systematically investigated the influence of NP properties-including size, shape, surface functionalization, and material-on electroporation efficacy. <b>Methods</b>: A combined approach using theoretical modeling and experimental validation was employed, encompassing numerical simulations, membrane permeabilization assays, transmission electron microscopy, and GET efficiency measurements. <b>Results</b>: Numerical results revealed that the presence of NPs alters local electric field distributions, but the amplification is highly localized, regardless of NP conductivity or geometry. Experimentally, only two out of six tested NP types produced a statistically significant, yet modest, increase in membrane permeability at one electric field intensity. Similarly, GET improvement was observed with only one NP type, with no dependence on concentration or functionalization. <b>Conclusions</b>: Overall, our findings demonstrate that NPs, under tested conditions, do not substantially enhance cell membrane permeability or GET efficacy. These conclusions are supported by both computational modeling and in vitro experiments.