Pulse intensity threshold regulates electroporation Nanodelivery: biological mechanisms and signaling Cascade

Abstract Background Reversible electroporation enhances nanoparticle internalization, but the coordinated regulation of delivery efficiency and cellular responses by pulse parameters remains unclear. This study aims to elucidate the regulatory logic underlying gradient electric pulse effects on nanoparticle uptake, cell survival, and a cascade of biological events—including oxidative stress, calcium influx, iron accumulation, and damage to mitochondria, lysosomes, and the endoplasmic reticulum—in A549 lung cancer cells. Results The pulse electric field intensity regulates the degree of cell membrane perforation at a threshold of 500–1000 V/cm. On the one hand, it gradually induces ion homeostasis disorder, oxidative stress amplification, multi organelle chain damage, and ultimately determines the fate bifurcation of cell survival. On the other hand, it synchronously and parallelly regulates the intracellular uptake efficiency of nanoparticles, forming a gradient regulation mainline that antagonizes the delivery effect and cell damage. Under square wave pulses (100 μs, 5 pulses, 1 Hz), 700 V/cm was validated as the critical inflection point within the 500–1000 V/cm RE‐to‐IRE transition interval for the RE safety window. Subthreshold stimulation generated slight biological disturbances and gradually improved delivery efficiency; Beyond this threshold, severe cell apoptosis can impair cell viability and delivery performance. The optimal concentration of nanoparticles has been confirmed to be 30 μg/mL. Conclusion This work defines an A549‐specific in‐vitro operational window for electroporation‐assisted nanoparticle delivery, uncovering threshold‐dependent biological effects. It offers in‐vitro reference parameters for tumor‐targeting platforms, supporting pulse‐protocol optimization as in‐vitro proof‐of‐concept for potential lung‐cancer strategies; clinical validation is still needed. © 2026 Society of Chemical Industry (SCI).