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-beta-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.
Kanatani
Isao
I
Department of Urology, Kyoto University Graduate School of Medicine, 54 Shogoinkawara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Ikai
Tomonori
T
Okazaki
Arimichi
A
Jo
Jun-Ichiro
J
Yamamoto
Masaya
M
Imamura
Masaaki
M
Kanematsu
Akihiro
A
Yamamoto
Shingo
S
Ito
Noriyuki
N
Ogawa
Osamu
O
Tabata
Yasuhiko
Y
D037263:Asialoglycoprotein Receptor / Q000235:genetics; D021941:Caveolae / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002966:Clathrin / Q000187:drug effects / Q000502:physiology*; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000235:genetics; D005936:Glucans / Q000737:chemistry*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013096:Spermine / Q000737:chemistry*; D014162:Transfection
D037263:Asialoglycoprotein Receptor; D002966:Clathrin; D004337:Drug Carriers; D005936:Glucans; C034477:RNA primers; D013096:Spermine; D012313:RNA; C009109:pullulan; D004247:DNA; D008156:Luciferases