Lacticaseibacillus paracasei–Derived Extracellular Vesicles Enhance Polyethyleneimine-Mediated Plasmid Transfection in HEK293 Cells via AKT Signaling

Non-viral gene delivery using polyethyleneimine (PEI) is limited by insufficient transfection efficiency and intracellular degradation of plasmid DNA. Here, we demonstrate that extracellular vesicles (EVs) derived from Lacticaseibacillus paracasei markedly enhance PEI-mediated plasmid transfection in HEK293 cells and clarify the underlying mechanisms. EVs increased reporter gene activity in a dose-dependent manner, independently of physical complex formation with PEI–plasmid DNA polyplexes. Quantitative analyses of intracellular plasmid DNA and mRNA levels revealed that EVs primarily promoted cellular uptake of plasmid DNA rather than transcriptional or translational efficiency. Pharmacological inhibition studies showed that EVs preferentially enhanced clathrin-mediated endocytosis. Fluorescence imaging further demonstrated reduced lysosomal colocalization of EV-associated signals, suggesting facilitated endosomal escape. Proteomic analysis identified the p40 protein on EVs, which associates with lipoteichoic acid (LTA), as a major EV-associated factor. Inhibition of Toll-like receptor 2 and platelet-activating factor receptor attenuated EV-mediated enhancement of reporter activity and suppressed AKT phosphorylation. Although epidermal growth factor–induced AKT activation increased reporter activity primarily through translational effects, EVs preferentially enhanced cellular uptake of plasmid DNA despite convergent AKT activation. Collectively, these findings demonstrate that L. paracasei-derived EVs enhance PEI-mediated gene delivery by promoting clathrin-dependent uptake of plasmid DNA, a process that appears to be initiated through p40–LTA-associated receptor signaling and AKT activation, thereby providing a mechanistic basis for EV-assisted non-viral gene delivery.