RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein

Nervous necrosis virus (NNV) can be transmitted vertically from parents to offspring, causing significant mortality in young fish. Therefore, effective decontamination of fertilized eggs is essential for sustainable aquaculture production. This study devel-oped siRNA-loaded PLGA nanoparticles (NPs) for targeted silencing of NNV capsid protein (CP) and evaluated their therapeutic potential in vitro. Using a dou-ble-emulsion method, siRNA-PLGA NPs were synthesized with optimized physico-chemical properties: the PLGA-PEI/trichloromethane/PEG (W/O/W) system yielded monodisperse, spherical nanoparticles (~ 110 nm post-sonication) with a high zeta po-tential (+ 30.91 mV), ensuring colloidal stability and efficient cellular delivery. Over-expression of NNV CP in EPC cells induced rapid cytotoxicity, including nuclear ab-normalities and cell death within 48 hours, which was fully rescued by co-administering three rationally designed CP-targeting siRNAs. Strikingly, nanopar-ticles resuspended in phosphate-buffered saline (PBS) exhibited a higher siRNA en-capsulation efficiency, whereas those suspended in Diethyl Pyrocarbonate water showed negligible loading. These findings underscore the importance of formulation parameters in nanoparticle design and highlight the promise of RNAi-based PLGA nanotherapeutics for combating aquatic viral infections.