Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 609

2.3 Endosomal Escape

The cellular endosome is a major barrier to efficient gene transfer. Endosome
escape mechanism is a necessary feature of nonviral complexes, especially targeted
vectors. Some peptides such as diphtheria toxin (DT) translocation domain under-
goes, melittin, and GALA peptide (mainly comprised of a Glu-Ala-Leu-Ala repeat)
can induce bending of the membrane and contiguity of the bilayer leaflets, thus
opening a pore in the membrane and facilitating release of endosomal contents
(Medina-Kauwe et al. 2005 ). Complexes conjugated with these functional peptides
can therefore escape endosome via the pore formation mechanism. For instance,
incorporation of melittin into PEI polyplexes led to 700-fold increase in gene trans-
fer of luciferase reporter gene (Ogris et al. 2001 ). The GALA mediated enhance-
ment of transfection was demonstrated by dendrimer-based vectors (Haensler and
Szoka 1993 ). Inspired by the endosomolytic capability of these peptides, an alterna-
tive class of polymers with specific alkyl groups has been developed. Among them,
poly(propylacrylic acid) was found to be the most promising one for gene delivery
(Murthy et al. 1999 ; Cheung et al. 2001 ).
For lipoplexes, it has been proposed a fusion and/or flip-flop mechanism for
endosomolysis (Xu and Szoka 1996 ). After their endocytosis, the lipoplexes
destabilize the membrane, inducing flipping of anionic lipids from the cytoplasm
to face the endosomal lumen. Then, the anionic lipids form charge neutral ion
pairs with cationic lipids of the complex, thus displacing the DNA and releasing it
into the cytoplasm. This mechanism was experimentally demonstrated by fluores-
cence resonance energy transfer (FRET) and fluorescence microscopy (Zelphati
and Szoka 1996 ).
A 'proton-sponge' mechanism has been attributed to the endosomal escape of
polyplexes based on cationic polymers such as polyethylenimine (PEI) and poly-
amidoamine (PAMAM) (Haensler and Szoka 1993 ; Boussif et al. 1995 ). Most of
the synthetic polymers used for gene delivery are polycations with amino groups.
These amino groups can be protonated during endosome acidification, thus offering
a high buffering capacity, commonly referred to as the 'proton-sponge' effect.
Massive proton accumulation results in high chloride ion influx into the endosome,
causing osmotic swelling and eventual endosomolysis and release of its contents
into the cytosol.

2.4 Cytosolic Transport and Nuclear Import

The cytoskeleton is thought to have dramatic effect on the intracellular trafficking
of endocytosed gene delivery complexes. Studies on lipoplexes based transfection
showed that microtubule disruption can enhance gene transfer level. By incorporat-
ing microtubule depolymerizing drugs (such as vinblastine) into lipoplexes, Wang
and MacDonald found approximately 30-fold increase in the transfection efficiency