Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 653

and Tenchov 2009 ). This hydrocarbon chain effect of cationic lipids is considered
to be related to the lipid phase properties as well as their interactions with mem-
brane lipids. A simple and direct correlation between phase structure and transfec-
tion activity has been proposed by some researchers. For instance, study by Zuhorn
et al. suggested that the formation of hexagonal phase in lipoplexes is important for
the cytoplasmic translocation of ODNs (Zuhorn et al. 2005 ). On the other hand,
Caracciolo et al. have demonstrated that DOTAP/DOPE/cholesterol liposomes with
a multilayer structure exhibit a more than 4-fold higher transfection activity in
OVCAR-3 and SK-OV-3 cells, compared with DC-Chol/DOPE liposomes with a
columnar inverted hexagonal phase (HII) (Caracciolo et al. 2003 ). Besides cationic
lipids, neutral lipids, such as dioleoylphosphatidylethanolamine (DOPE) and dio-
leoyl phosphatidyl choline (DOPC) are required to stabilize the CL suspension, to
facilitate membrane fusion and endosomal escape (Felgner et al. 1994 ; Zuidam and
Barenholz 1998 ; Hafez et al. 2001 ). More often, the transfection activity of lipo-
plexes may be impaired if they are formulated without neutral lipid(s) (Felgner
et al. 1994 ; Mui et al. 2000 ). It should be noted that the interactions of lipoplexes
with cellular membrane may transform the original structure, rendering a new
structural organization. Consequently, the transgene efficacy of lipoplexes depends
not only on the formulations of CLs and their structure, but also on their interac-
tions with cell membrane and the resulting structures (de Ilarduya et al. 2010 ).
Strategies employed to improve the lipoplexes-based gene delivery in vivo
include enhancing the stability and circulation time in bloodstream, targeting to
specific tissues and cells, and facilitating intracellular trafficking. To prolong the
circulation time of lipoplexes, PEG-conjugated lipids can be incorporated into the
CL (Fenske et al. 2002 ). Additionally, PEGylation of CLs may reduce the toxicity
and stabilize lipoplexes in vivo . However, PEG-lipid substantially decreases the
intracellular uptake of lipoplexes and impedes intracellular trafficking to some
degree, and therefore reduces gene transfection efficiency. To partly circumvent this
problem, Szoka's group synthesized an acid-labile PEG-diorthoester-lipid conju-
gate (POD) (Guo and Szoka 2001 ), in which the diorthoester linkage may be rap-
idly cleaved as pH decreases from 7.0 to 5.0 (pH value in the endosome). They also
demonstrated that the lipoplexes with pH-sensitivity displayed better in vitro trans-
fection activity than the pH-insensitive counterparts (Choi et al. 2003 ). Alternatively,
novel lipids bearing acid-labile linkages, such as vinyl ether (Boomer et al. 2002 ),
ketal (Zhu et al. 2002 ), ortho ester (Zhu et al. 2000 ), or acylhydrazone (Mueller
et al. 1990 ), may be employed to construct pH-sensitive lipoplexes with improve
gene transfer performance. Redox potential-sensitive lipids are another family of
triggerable vectors, and disulfide is the most frequently used redox-sensitive linker.
Table 8 shows the representative lipids with disulfide linkage. For example, lipo-
plexes based on disulfide-containing lipid DOGSDSO showed 6-15-fold or 50-fold
transfection efficiency compared with those based on DOTAP or a non-cleavable
analogue, respectively (Tang and Hughes 1998 ).
A common problem for the cationic lipids-based vectors is their toxicity, par-
ticularly in cultured cells (Dass and Choong 2006 ). The lipoplexes related toxicity
has also been observed in animal studies and even in human patients at higher doses