Mechanism of cell transfection with plasmid/chitosan complexes.
Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.
Ishii
T
T
Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan.
Okahata
Y
Y
Sato
T
T
D001798:Blood Proteins; D002460:Cell Line; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D048271:Chitosan; D015536:Down-Regulation; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008970:Molecular Weight; D009584:Nitrogen / Q000032:analysis; D010316:Particle Size; D010710:Phosphates / Q000032:analysis; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D013501:Surface-Active Agents; D014162:Transfection; D014966:Xanthenes
D001798:Blood Proteins; D005456:Fluorescent Dyes; D010710:Phosphates; D011522:Protons; D013501:Surface-Active Agents; D014966:Xanthenes; D002686:Chitin; C034657:Texas red; D048271:Chitosan; D016650:Fluorescein-5-isothiocyanate; D009584:Nitrogen