Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 63

ionizable end groups. After the arrival of PEI-DNA polyplexes in the endosomes, it
becomes protonated at an acidic pH of 5.5-6.0, causing proton flux into the endo-
somal lumen by the Donnan effect [135,136] and simultaneously pulls chloride ions
and water in as well. The proton sponge hypothesis assumes that PEI, which has been
shown to be only 11% protonated at pH 7.5 in water [137] , can effectively buffer the
pH of the endosomal compartment. This buffering effect results in an increased flux of
protons (and hence chloride ions and water) into the endosome, which causes swell-
ing and, ultimately, osmotic lysis of the endosome [133] . Significantly, complexation
of PEI with negatively charged DNA further neutralizes the positive charge and thus
shifts the p K a of PEI amines, thereby resulting in significantly enhanced degree of
protonation [132,136,137] .
Behr et al. also observed that, because of the high density of positive charges
and bifurcated structures, these substances allow efficient binding of DNA. They
are advantageous over the quaternary amine-containing lipids such as DOTAP, DC-
Chol, and DOTMA because these lipids are unable to cause the proton sponge effect
and because of the weak buffering capacity, a result of less charge density [133] .
Therefore, high molar fractions of helper lipids such as DOPE are required with the
cationic lipids.
However, contradictory observations have also been observed, suggesting re-eval-
uation of the proton sponge hypothesis. The Donnan effect is observed to be signifi-
cantly reduced at increased ionic strength [135,136] . Further, it was observed that, in
the presence of PEI, no substantial differences in lysosomal pH of treated cells are
observed compared to untreated cells, suggesting an inability of PEI to substantially
buffer these compartments [138] . However, the pH-dependent release of PEI-DNA
complexes is also supported by the ability of ionophores, which reduce the trans-
membrane pH gradient, to significantly inhibit transgene expression [132] .
To enhance the endosomal release of DNA from cationic lipids, apart from the
helper lipids, additional helper substances have been used such as chloroquine, viral
fusogenic peptides, and pH-sensitive polymers.
Chloroquine, the most commonly used endoosmolytic agent, inhibits the lysosomal
processing and thus delays degradation of DNA in the lysosome [139] . Use of osmotic
agents such as glycerol, sucrose, or PVP at higher concentration has also been found
to increase transgene expression, presumably by causing osmotic lysis of endosomes
[139-141] . The use of viral fusogenic peptides with pH-dependent membrane lytic
activity has also been shown to enhance endosomal escape of DNA [103,142] . The
synthetic peptides mimicking the 20 amino-terminal amino acid sequence of influenza
virus HA [143] was found to destabilize the endosomal membrane in an acidifica-
tion-dependent manner similar to that exhibited by the viruses. When transferrin-poly
(L-lysine)-DNA complex was mixed with these negatively charged fusogenic peptides,
they were bound by the ionic interactions. These peptides have shown remarkable
enhancement in their transfection capacity in human melanoma cells [144] and have
also been effective under in vivo conditions. The peptide mHA2 from the influenza
virus, after conjugating to galactosylated poly(L-ornithine), enhanced plasmid delivery
to liver parenchymal cells selectively (95%) by two fold. The confocal studies per-
formed in vitro in HepG2 cells revealed distribution of fluorescent-labeled DNA in