Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 64

cytosol after using the mHA2 as compared to localization of DNA in lysosome and
endosome in the cells without the mHA2 treated vector [145] .
The pH-sensitive polymers such as poly(L-histidine) have also shown a prop-
erty of inducing membrane fusion at endosomal pH values and have been used
along with poly(L-lysine) to form a graft copolymer N -Ac-poly(L-histidine)- graft -
poly(L-lysine) with higher transfection efficacy in 293T cells than PLL at all equiv-
alent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic
agent further enhanced transfection for both PLL and PLH- g -PLL gene carriers in
the cell line [146] . Another pH-sensitive anionic polymer, poly(propylacrylic acid)
(PPAA), has also shown improved transfection efficiencies in NIH3T3 fibroblast
cells when used along with cationic lipid DOTAP and pCMV plasmid DNA. PPAA
also significantly improved the serum stability of DOTAP/DNA vectors [147] .
After avoidance of lysosomal degradation by the endosomal escape of the DNA-
polycation complex, the release of DNA from the DNA-polycation complex is an
essential step for efficient transgene expression. The release of DNA from cationic
lipoplex or polyplex may occur during escape from endosomal membranes, in cytosol
or in nucleus. It has been evidenced that the dissociation of cationic lipids from the
DNA precedes the nuclear uptake of the complex via a nuclear pore complex (NPC)
[148] . The study was conducted to demonstrate that microinjection of DNA alone
produces more transgene expression than the DNA-lipid complex inside the nucleus
[149,150] . The release of DNA from the complex may be facilitated by the presence
of polyanions, such as negatively charged phosphatidylserine, in the endosomal mem-
brane [127,128] , and RNA in the cytosol. Further, a study indicated dissociation of
polycation-plasmid DNA in the cytosol, as demonstrated by the T7 polymerase trans-
fection system that permits the transcription of a reporter gene controlled by the T7
promoter in the cytoplasm [151] . However, the recent localization experiments with
PEI-DNA or PLL-DNA complex have shown significant transgene expression when
compared with the naked DNA and DNA-cationic lipid complex, after direct micro-
injection into the nucleus, which suggests possible dissociation of PEI from DNA
inside nucleus [150,152,153] .
Still, the productive transcription-translation of cytoplasmic plasmid DNA
demonstrated exposure of a small fraction of DNA to the cytosol during its cellu-
lar transport and presents further barriers to the transgene expression because of the
physicochemical and biological properties of the cytosol.
2.4.2 Cytosolic Transport of DNA
The cytoplasm is composed of a network of microfilament and microtubule systems
with the presence of subcellular organelles. Cytoplasm consists of a meshlike struc-
ture because of the presence of actin filaments, microtubules, and intermediate fila-
ments. These filaments, together with actin- and tubulin-binding proteins and several
enzymes, constitute the cytoplasm and maintain the cell structure [154] . The meshlike
structure of the cytoskeleton, the presence of organelles, and the high protein concen-
tration (up to 100 mg/ml) impose significant molecular crowding of the cytoplasm,
which limits the diffusion of large-sized macromolecules in the cytoplasm [154] .