Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking.
We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.
Joshee
Nirmal
N
Department of Biochemistry and Molecular Biology, College of Medicine, and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-4525, USA.
Bastola
Dhundy R
DR
Cheng
Pi-Wan
PW
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D018014:Gene Transfer Techniques; D006046:Gold / Q000494:pharmacology; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010714:Phosphatidylethanolamines / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014966:Xanthenes / Q000494:pharmacology; D001616:beta-Galactosidase / Q000378:metabolism
D010714:Phosphatidylethanolamines; D014168:Transferrin; D014966:Xanthenes; D006046:Gold; C020888:1,2-dielaidoylphosphatidylethanolamine; C034657:Texas red; D004247:DNA; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase