Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles

Abstract

This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40–200 nm as determined using photon correlation spectroscopy (PCS) and 40–80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding β-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect™ and Lipofectin™ were also used. In contrast to chitosan particles, the duration of expression for both SuperFect™ and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect™ and Lipofectin however, were observed to engender marked cytotoxicity. Poly- d, l-lactide (PLA) nanoparticles (40–80 nm) and poly- l-lactide (PLLA) lamellae (2–6 μm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.