Immersion delivery of plasmid DNA. II. A study of the potentials of a chitosan based delivery system in rainbow trout (Oncorhynchus mykiss) fry.

The possibility of utilising DNA vaccines for aquaculture fish has been of growing interest in recent years and novel methods to deliver DNA to the fish are under investigation. One of the delivery methods of interest is immersion. Due to the favourable properties of chitosan in gene delivery and bioadhesion, chitosan-DNA formulations have been investigated for use for immersion delivery to fish. Initial studies on this system, however, revealed an acute toxic effect of the formulations. In this study, factors important for the acute toxicity of chitosan and chitosan formulations are identified and attempts are made to explain the underlying mechanisms for the toxicity. In vivo methods revealed that the toxicity mainly was dependent on the concentration of chitosan, but also the molecular weight and the degree of acetylation of the chitosans were of importance. Noteworthy, the toxicity of the polymer decreased dramatically when the chitosan was 'decharged' by complexation with DNA. In vitro experiments supported the in vivo observations. Most likely, the observed toxicity is caused by an electrostatic interaction between the cationic polymer and the anionic parts of the gill mucus. The result is obstructed oxygen diffusion over the gills and the fish are killed by acute hypoxia. Careful selection of chitosan type and charge of the particles may result in a potential for chitosan based immersion delivery of plasmid DNA.