RESEARCH ARTICLE
Atomic force microscopy imaging of DNA±cationic
liposome complexes optimised for gene
transfection into neuronal cells
Louise A. Wangerek
1,2
Hans-Henrik M. Dahl
1,2
Tim J. Senden
3
John B. Carlin
2,4
David A. Jans
5
Dave E. Dunstan
6
Panayiotis A. Ioannou
1,2
Robert Williamson
1,2
Susan M. Forrest
1,2
*
1
Murdoch Children's Research
Institute, Royal Children's Hospital,
Parkville, Australia
2
Department of Paediatrics,
University of Melbourne, Royal
Children's Hospital, Parkville,
Australia
3
Department of Applied Mathematics,
Research School of Physical Sciences
and Engineering, Australian National
University, Canberra, Australia
4
Clinical Epidemiology and
Biostatistics Unit, Murdoch Children's
Research Institute, Parkville,
Australia
5
Nuclear Signalling Laboratory,
Division for Biochemistry and
Molecular Biology, John Curtin
School of Medical Research,
Australian National University,
Canberra, Australia
6
Department of Chemical
Engineering, University of Melbourne,
Parkville, Australia
*Correspondence to: Susan M.
Forrest, Murdoch Children's
Research Institute, Royal Children's
Hospital, Flemington Road,
Parkville, 3052 VIC, Australia.
E-mail:
forrest@cryptic.rch.unimelb.edu.au
Received: 5 June 2000
Revised: 3 October 2000
Accepted: 5 October 2000
Abstract
Background Cationic liposomes represent an important gene delivery
system due to their low immunogenicity, but are relatively inef®cient, 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 ef®ciency.
Methods Four commercially available cationic liposome formulations were
used to optimise transfection ef®ciency in neuronal cells. The DNA to
liposome ratio and the amount of DNA used in transfections were varied.
Transfection ef®ciency was determined by the percentage of cells positive for
the b-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 ¯uorescence techniques
to show DNA compaction.
Results Optimal transfection conditions were found to differ between
immortalised cell lines and primary cells. High transfection ef®ciencies in
immortalised cell lines were achieved predominantly with multivalent
cationic liposomes while primary neuronal cells showed optimal transfection
ef®ciency 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 ®nding, high levels of DNA compaction
with multivalent liposomes were observed using ¯uorescence 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 ef®ciency was different for cell
lines and primary neurons. Immortalised cells showed optimal transfection
with multivalent liposomes while primary neurons showed optimal transfec-
tion 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
THE JOURNAL OF GENE MEDICINE
J Gene Med 2001; 3: 72±81.
Copyright # 2001 John Wiley & Sons, Ltd.