Feed-based delivery of plasmid DNA using milk-derived extracellular vesicle-polyethylenimine complexes enables hepatic transgene expression in goldfish

Oral administration through feed offers a non-invasive, cost-effective, and scalable approach for gene delivery in aquaculture. However, gastrointestinal degradation and intestinal barriers severely limit the oral delivery of plasmid DNA (pDNA). In this study, we developed a ternary nanocarrier composed of bovine milk-derived extracellular vesicles (MEVs), polyethylenimine (PEI), and pDNA (MPPs) to investigate its potential for functional gene delivery to the goldfish (Carassius auratus) liver (hepatopancreas). The resulting MPPs exhibited a relatively uniform particle size, high pDNA loading efficiency, and protection against nuclease-mediated degradation. Formulation optimization in HEK293T cells identified an MEV:PEI:pDNA mass ratio of 16.7:4.1:1, which supported robust EGFP reporter expression. Following oral administration of DiD-labeled MEVs through feed, fluorescence signals were detected in the goldfish hepatopancreas within 2 h and remained detectable at 6 h. After a single feeding of MPP-containing feed, hepatic luciferase mRNA expression peaked at 24 h and gradually declined to near-baseline levels by 72 h. During 14 consecutive days of feeding, luciferase protein expression was detectable from day 3, with progressively increased protein abundance and enzymatic activity observed on days 7 and 14. Collectively, these findings demonstrate the feasibility of feed-based MPP administration for inducing functional transgene expression in the goldfish hepatopancreas, providing a promising foundation for developing non-invasive nucleic acid delivery strategies in aquaculture.