Efficient gene transfer by pullulan–spermine occurs through both clathrin- and raft/caveolae-dependent mechanisms

The feasibility and mechanism of gene delivery by pullulan–spermine, a recently developed cationic polysaccharide, were investigated. Pullulan–spermine-mediated transfection of plasmid DNA resulted in greatly reduced cytotoxicity and a 10-fold increase in the level of gene expression when compared to Lipofectamine 2000, a commercially available cationic lipid. Additionally, after transfection of p53-expressing plasmid DNA by pullulan–spermine but not Lipofectamine 2000, the in vitro proliferation of T24 cells was significantly reduced. Pullulan–spermine-mediated gene expression was inhibited by both chlorpromazine of clathrin-mediated endocytosis inhibitor and methyl-β-cyclodextrin and filipin of raft/caveolae inhibitors. We conclude that pullulan–spermine is a promising carrier for gene transfection, and that cellular uptake of pullulan–spermine–plasmid DNA complexes is mediated by clathrin- and raft/caveolae-dependent endocytotic pathways.