Nanoliposomes encapsulating immunostimulants modulate the innate immune system and elicit protection in zebrafish larvae

Here we present immunostimulant-loaded nanoliposomes (NLc) as a strategy to protect zebrafish larvae against bacterial infection. The NLc encapsulate crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid (Poly I:C), a synthetic analogue of viral dsRNA. Fluorescently-labeled NLc were ingested by zebrafish larvae 4 days post fertilization, when administrated by bath immersion, and accumulated in ;the intestine. RT-qPCR analysis showed the expression of innate immune related genes (tnf alpha, ill beta, nos2a, irfla and ptgs2a) was significantly upregulated at 48 h post NLc, treatment. A zebrafish larvae infection model for Aeromonas hydrophila was set up by bath immersion, achieving bacterial-dose-dependent significant differences in survival at day 5 post infection in both injured and non-injured larvae. Using this model, NI., protected non injured zebrafish larvae against an A. hydrophila lethal infection. In contrast, neither the empty nanoliposomes nor the mixture of immunostimulants could protect larvae against lethal challenges. Our results demonstrate that nanoliposomes could be further developed as an efficient carrier, widening the scope for delivery of other immunostimulants in aquaculture.