Atomic force microscopy imaging of DNA-cationic liposome complexes optimised for gene transfection into neuronal cells.
BACKGROUND
Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency.


METHODS
Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the micro-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction.


RESULTS
Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied.


CONCLUSIONS
Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as
Wangerek
L A
LA
Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia.
Dahl
H H
HH
Senden
T J
TJ
Carlin
J B
JB
Jans
D A
DA
Dunstan
D E
DE
Ioannou
P A
PA
Williamson
R
R
Forrest
S M
SM
D000818:Animals; D016466:CHO Cells; D002412:Cations; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D008027:Light; D008081:Liposomes; D018625:Microscopy, Atomic Force; D009474:Neurons / Q000378:metabolism*; D012542:Scattering, Radiation; D014162:Transfection
D002412:Cations; D008081:Liposomes; D004247:DNA