Intracellular Delivery Considerations for RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 85

Langer et al. used titratable pH-sensitive lipids to design polymer-protected
cationic liposomes with minimized binding of serum proteins. The liposomes were
then able to shed the protecting polymer and restore their fusogenic ability after
internalization into the acidic environment in the endosomes [ 77 ] . Carmona et al.
developed liposomes stabilized by PEG attached to cholesterol by pH-sensitive
oxime linkage, whose hydrolysis then facilitates pH-triggered escape of the lipo-
somes from the endosomes [ 41 ]. Andaloussi et al. utilized pH titratable
trifluoromethylquinoline moieties and covalently linked them to CPP, which resulted
in rapid endosomal release [ 42 ] .
Pluronic block copolymers exhibit a thermally reversible swelling/deswelling
behavior, which triggers endosomal escape of RNAi delivery vectors. Nanocapsules
prepared from PEI and Pluronic were able to efficiently deliver siRNA, in part, due
to improved endosomal escape [ 43 ] .
Photosensitive vectors utilize photosensitizers for endosomal disruption. Upon
exposure to light, photosensitizers are stimulated to form reactive oxygen species
leading to the damage of endosomal membranes, causing release of the RNAi vec-
tors in the cytoplasm. Oliveira et al. have shown that by incorporating photosensi-
tizers into siRNA delivery vectors which target epidermal growth factor receptor
(EGFR), endosomal escape efficiency of siRNA vectors improved significantly,
resulting in a tenfold increase in the knockdown efficacy of EGFR [ 44 ] .

4.3.2.3 Fusogenic Lipids and Peptides

Lipid vectors with non-lamellar structure adhere to anionic vesicles without induc-
ing fusion event [ 68 ]. In contrast, lipids with inverted hexagonal structure fuse with
anionic membranes and cause the release of nucleic acids from endosomes [ 78 ] .
Thus, incorporation of fusogenic lipids such as 1,2-dioleoyl-sn-glycero-3-phos-
phoethanolamine (DOPE) into the delivery vectors enhances endosomal escape of
siRNA [ 45 ] .
An alternative to fusogenic lipids is to use short peptides that mimic the fusion
process of viruses with endosomal membranes during infection. Several synthetic
fusogenic peptides mimicking the fusion domain of the influenza virus have been
developed. Oliveira et al. reported that by incorporating influenza-derived fuso-
genic peptide diINF-7 into the delivery system, gene silencing efficiency of siRNA
targeting EGFR and K-ras oncogenes is significantly enhanced [ 46 ] .
Another alternative approach is to introduce pore-forming ability of viroporins
into the delivery vectors. Viroporins are a group of hydrophobic proteins that par-
ticipate in several steps during viral infection, including the release of viral particles
from cells, altering membrane permeability, creating channels, and facilitating ion
fl ow across membranes [ 79 ]. Kwon and coworkers have chosen a membranolytic
peptide from the endodomain of HIV gp41 and covalently linked it to PEI. The
peptide-modified polyplexes exhibited significantly enhanced siRNA delivery
efficiency compared to unmodified control polyplexes. Confocal microscopy imag-
ing of intracellular polyplex distribution suggested that the enhanced transfection
was the result of increased endosomal escape [ 47 ] .