Nanosecond Pulsed Electric Fields for Extracellular Vesicle Engineering: From Electro-Exocytosis to Cargo Modulation

Extracellular vesicles (EVs), including small extracellular vesicles (sEVs) and medium/large extracellular vesicles (MVs), have emerged as promising therapeutic vectors and diagnostic biomarkers across various branches of biomedicine. However, the clinical translation of EV-based technologies remains constrained by persistent challenges in manufacturing: insufficient yield from primary cell sources, limited control over cargo composition, and the absence of scalable, standardized production platforms. Nanosecond pulsed electric fields (nsPEF) represent an emerging biophysical approach that can address several of these limitations. Unlike conventional electroporation, which targets the plasma membrane using microsecond-to-millisecond pulses, nsPEF delivers ultrashort (1–300 ns), high-amplitude (10–300 kV/cm) pulses that penetrate intracellularly to directly perturb endosomal membranes, the endoplasmic reticulum, and the multivesicular body (MVB) compartment, the very organelles where small EVs (exosome) biogenesis and cargo sorting occur. Through coordinated effects on intracellular calcium mobilization, cytoskeletal remodeling, SNARE-mediated membrane fusion, and phospholipid redistribution, nsPEF can stimulate rapid, non-lethal vesicle release, a process labeled as “electro-exocytosis.” Emerging and growing evidence suggests that nsPEF does not merely increase EV yield but actively modulates the proteomic, lipidomic, and nucleic acid composition of released vesicles, offering a potential route to cargo engineering. In addition, the same biophysical principles that drive electro-exocytosis can be exploited in reverse: nsPEF-mediated transient permeabilization of EV membranes allows for post-isolation loading of exogenous therapeutic cargo, small molecules, nucleic acids, or proteins into pre-formed vesicles without destroying their structural integrity. This review discusses current knowledge on EV biogenesis and release mechanisms, introduces the biophysical foundations of nsPEF–cell and nsPEF–membrane interactions, and, by evaluating the experimental evidence supporting nsPEF-driven EV engineering, outlines a translational roadmap for the application and development of this technology toward clinical-grade EV manufacturing.