A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Abstract

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.

Zebrafish liver (ZFL) cells are able to mount an anti-viral response after stimulation with Poly (I:C)

“…Both genes have been described as interferon inducible genes. Also, MXa is an interferoninduced GTP-binding protein with direct antiviral activity and well described to assess viral response due to its high and consistent expression levels (Altmann et al, 2004;Verrier et al, 2011;Ruyra et al, 2013). By comparing the expression of MXa and GIG2 in ZFL cells stimulated with Poly (I:C) (PCR and microarray data), to their expression in adult zebrafish livers after Poly (I:C) i.p.…”

Relation between biophysical properties of nanostructures and their toxicity on zebrafish

“…therapy in mice and observed no side effects. In the same line, Ruyra et al (2013) introduced an immunostimulant cocktail in liposomes and studied toxicity in zebrafish embryos and larvae by evaluating the hatching rate, mortality and malformations. They found that embryos were able to hatch and develop normally and observed no morphological defects.…”

“…They studied the in vivo toxicity of cationic liposomes in different formulations, and observed that toxicity was affected by the dose and the concentration of encapsulated lipopolysaccharide in the lipidic bilayer of liposomes. When comparing the survival between embryos and larvae, Ruyra et al (2013) found that the mortality in larvae was higher. Although not discussed by the authors, the difference may be due to the presence of the chorion in the embryo, which acts as a protective barrier.…”

Molecular transfer to Atlantic salmon ovulated eggs using liposomes

“…Several studies in higher vertebrates have shown that liposome surface charge has a profound effect on the liposome-cell interaction in vitro (Cho et al, 2009;Düzgüneş and Nir, 1999;Miller et al, 1998). Liposomal surface charge and applied concentrations have an effect on the liposome-cell interaction as well as the cytotoxicity as confirmed in the teleost (zebra fish and trout) studies (Romøren et al, 2005;Ruyra et al, 2013). Based on the similar findings regarding cytotoxicity of positively charged liposomes in fish and mammals, only neutral and negatively charged liposomes were used in the present research focused on the preliminary uptake study.…”

“…In aquaculture, liposomes have been, up to now, mostly used as carriers for zooplankton nutrients enrichment (Barr and Helland, 2007;Hawkyard et al, 2015) in bivalve mollusc larvae (Venerupis decussatus and Venerupis pullastra) (Lai et al, 2004) and therapeutic agents, such as immunostimulants and vaccines (Ji et al, 2015;Ruyra et al, 2013). For example, liposomes with lipopolysaccharides (LPS) from Aeromonas salmonicida gave rainbow trout better protection against furunculosis than the LPS administered alone (Nakhla et al, 1997).…”