Synthesis and characterization of CS/TPP nanoparticles for oral delivery of gene in fish

Abstract

This work examines the potential use of chitosan tripolyphosphate (CS/TPP) nanoparticles for gene delivery in different tissues of fish through oral route. The porin gene of Vibrio anguillarum was used to construct a DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP. The CS/TPP nanoparticles were synthesized by ionic gelation process and these nanoparticles particles were characterized. The morphology and particle size measurements of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The pVAOMP was encapsulated by CS/TPP nanoparticles by mixing of equal volume of heated CS/TPP and pVAOMP DNA solutions. The encapsulation efficiency of CS/TPP nanoparticles was found to be 79.9% of DNA binding with CS/TPP nanoparticles. The stability of plasmid DNA was also determined after encapsulation using DNase I and chitosanase. The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by immunohistochemistry, ELISA and real-time PCR analyses. The results indicate that DNA can be easily delivered into fish by feeding with CS/TPP nanoparticles.