A novel low-molecular-weight chitosan/gamma-polyglutamic acid polyplexes for nucleic acid delivery into zebrafish larvae.

Many challenges, such as virus infection, extreme weather and long cultivation periods, during the development of fish larvae have been observed, especially in aquaculture. Gene delivery is a useful method to express functional genes to defend against these challengers. However, the methods for fish larvae are insufficient. In our earlier report, low-molecular-weight chitosan (LMWCS) showed a strong positive charge and may be useful for polyplex formulation. Herein, we present a simple self-assembly of LMWCS polyplexes (LMWCSrNPs) for gene delivery into zebrafish larvae. Different weight ratios of LMWCS/gamma-polyglutamic acid (γ-PGA)/plasmid DNA were analyzed by gel mobility assay. Delivery efficiency determined by green fluorescent protein (GFP) expression in zebrafish liver (ZFL) cells showed that delivery efficiency at a weight ratio of 20:8:1 was higher than others. Zeta potential and transmission electron microscopy (TEM) analysis showed that the round shape of the particle size varied. In our earlier reports, IRF9S2C could induce interferon-stimulated gene (ISG) expression to induce innate immunity in zebrafish and pufferfish. Further delivery of pcDNA3-IRF9S2C-HA plasmid DNA into ZFL cells and zebrafish larvae by LMWCSrNP successfully induced ISG expression. Collectively, LMWCSrNP could be a novel gene delivery system for zebrafish larvae and might be used to improve applications in aquaculture.