Cationic cholesterol promotes gene transfection using the nuclear localization signal in protamine.
OBJECTIVE
The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin).


METHODS
Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37 degrees C. The cells then were washed and cultured for another 40 h in the growth medium at 37 degrees C before luciferase assay.


RESULTS
The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine.


CONCLUSIONS
Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.
Noguchi
Ari
A
Graduate School of Pharmaceutical Sciences. Nagoya City University, Nagoya, Japan.
Hirashima
Naohide
N
Nakanishi
Mamoru
M
D016475:3T3 Cells; D000818:Animals; D002412:Cations / Q000008:administration & dosage / Q000737:chemistry; D002784:Cholesterol / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D019913:Nuclear Localization Signals / Q000008:administration & dosage / Q000235:genetics*; D011479:Protamines / Q000008:administration & dosage / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D002412:Cations; D019913:Nuclear Localization Signals; D011479:Protamines; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D002784:Cholesterol