68Biochemical Society Transactions (2007) Volume 35, part 1
Gene delivery by cationic lipids: in and out
of an endosome
D. Hoekstra1, J. Rejman2, L. Wasungu, F. Shi3and I. Zuhorn
Department of Cell Biology, Section Membrane Cell Biology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1,
9713 AV Groningen, The Netherlands
Cationic lipids are exploited as vectors (‘lipoplexes’) for delivering nucleic acids, including genes, into cells
for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy
needs major improvement, and better defining the mechanism of entry in relation to eventual transfection
efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative
contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or
macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought
to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes,
facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal
membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery.
To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-
mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems
The ability of introducing and expressing genes into cells
and cell biological exploitation of this technology [1–5].
of genetic origin by introducing the defective gene(s) in mal-
possibility of investigating biological properties of expressed
protein(s). Since DNA molecules are internalized poorly by
cells, a crucial aspect of this technology entails the efficiency
currently being exploited, including virus-derived and non-
viral vehicles, such as synthetically prepared cationic lipids
(Figure 1) and polymers.
Although non-viral-system-mediated transfection is still
inferior compared with viral systems, safety hazards en-
genic side effects, justify research to improve non-viral trans-
fection efficiency. Part of the approach relies on the synthesis
of novel cationic lipids and polymers. However, given cur-
rent insight into how cationic lipid–DNA (‘lipoplexes’) or
polymer–DNA complexes (‘polyplexes’) transfect cells, it
seems equally useful to further explore mechanisms of gene
delivery to better appreciate and overcome potential hurdles
in overall transfection, which range from complex assembly
to intracellular trafficking and transcription efficiency. For
Key words: cationic lipid, endocytosis, gene delivery, hexagonal phase, lipoplex, transfection.
Abbreviations used: DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PEG, poly(ethyl-
ene glycol); PS, 1,2-acyl-sn-glycero-3(phospho-l-serine).
1To whom correspondence should be addressed (email firstname.lastname@example.org).
2Present address: Institute for Experimental Treatment of Cystic Fibrosis, San Rafaella Scientific
Institute, Milano, Italy.
3Present address: Department of Otology and Laryngology, Harvard Medical School, Boston,
MA 02114, U.S.A.
example, such studies may prompt the rational develop-
ment of lipoplexes, containing properly complexed and tran-
scription-competent DNA, targeted into those intracellular
delivery efficiency, is essential, and recent work suggests
that a precise quantification of DNA delivery, including dis-
sociation from the carrier and eventual transcription effi-
ciency, is necessary for the proper appreciation of barriers
in gene delivery . Therefore in vitro experiments using
are not necessarily sufficient to warrant in vivo success [1,7].
Thus additional barriers exist in vivo that are insufficiently
preliminary clearance of DNA vectors by macrophages and
to achieve targeting to desired tissue and/or cells. To avoid
non-specific binding in vivo of the usually net-positively
charged lipoplexes, PEG [poly(ethylene glycol)]-derivatized
properties conveyed in this manner may require a proper
we will briefly discuss some recent developments in ad-
vancing our understanding of the mechanisms of lipoplex-
mediated transfection, with an emphasis on lipoplex internal-
ization and trafficking, leading to expulsion of DNA into the
cytosol as a prerequisite for nuclear gene delivery. For more
detailed recent overviews, see [3,5,10–13].
Assembly and phase behaviour
Liposomes prepared from cationic amphiphiles interact with
C ?2007 Biochemical Society