In vitro gene delivery mediated by chitosan. Effect of pH, serum, and molecular mass of chitosan on the transfection efficiency

Abstract

Aminopolysaccharides such as chitosan and polygalactosamine (pGalN) were used to transfer luciferase plasmid into tumor cells. Chitosan largely enhanced the transfection efficiency of luciferase plasmid (pGL3), while pGalN did not at all. Transfection efficiencies of the pGL3/chitosan complexes were dependent on pH of culture medium, stoicheometry of pGL3 : chitosan, serum, and molecular mass of chitosan. Transfection efficiency at pH 6.9 was higher than that at pH7.6. Optimum charge ratio of the pGL3 : chitosan was 1 : 5. Chitosan polymer of 15 and 52 kDa largely promoted luciferase activities. Transfection efficiency mediated by chitosan of >100 kDa was less than that by chitosan of 15 and 52 kDa. Heptamer (1.3 kDa) did not show any gene expression. Cationic liposome (lipofectin)-associated gene expression was inhibited by serum, while chitosan showed resistance to serum.

Introduction

Recently, a number of new techniques have been developed to introduce a foreign DNA into cells. One approach is non-viral delivery system based on supramolcular assembly. Cationic lipids [1], [2], [3], [4], [5], [6], [7] and cationic polymers [8] have been employed as non-viral gene transfer agents. These cationic substances form complexes with anionic DNA by electrostatic interaction. The cationic DNA complexes obtained were uptaken by cells through the electrostatic interaction because cell surface is negatively charged. Transfection efficiencies of these complexes have been investigated in vivo and in vitro. However, there are several problems to be overcome before practical use. Those involve insufficient transfection efficiency, a strong cytotoxicity, and inhibition by serum.

Chitosan is a linear cationic polysaccharide composed of β1→4 linked glucosamine partly containing N-acetylglucosamine. Chitosan is known to be biodegradable polymer [9]. Thus, chitosan microspheres have been employed as drug-delivery system [10], [11], [12]. In our previous paper, we prepared the DNA/chitosan and DNA/polygalactosamine (pGalN, α1→4 linked galactosamine) complexes, and investigated the interaction of the DNA complexes with tumor cells and white blood cells [13]. Especially, the DNA/chitosan complex was uptaken by tumor cells, but not by white blood cells. After our report, chitosan (1000–2000 kDa)-mediated transfection was preliminary assessed by using β-galactosidase plasmid in the serum-free medium [14]. Thereafter, transfection ability of luciferase plasmid mediated by chitosan (70 kDa) and lactose-conjugated chitosan was investigated using several cell lines [15]. Furthermore, Fiona et al. [16] and Roy et al. [17] reported in vivo plasmid delivery mediated by chitosan. As shown in these papers [14], [15], [16], [17], in vitro and in vivo gene delivery mediated by chitosan has been successful. Thus, chitosan is one of candidates for practical gene carrier. However, optimum condition for the transfection efficiency of DNA/chitosan complex has not been clear. In general, biological functions of polymer are largely dependent on the molecular mass and experimental condition of culture medium (pH or additives). It is considered that gene-transfer efficiency promoted by cationic polymer will be inevitably modulated by the characteristics of polymer. In this report, we investigated the effects of experimental conditions such as pH of medium, serum, and molecular mass of chitosan on the transfection efficiency of plasmid/chitosan complexes. We found that the transfection efficiencies of the luciferase plasmid (pGL3)/chitosan complexes were enhanced in culture medium of pH 6.9 and in the presence of serum, when the molecular mass of chitosan was 15–52 kDa. Furthermore, chitosan showed higher gene transfer efficiencies at optimum conditions than lipofectin and pGalN did.