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endogenous enhanced green fluorescent protein in H1299 human lung carcinoma
cells and murine peritoneal macrophages (78% and 89% knockdown of the
fl uorescence signal, respectively) [ 54 ] .
4.3.5.2
Reductively Degradable Delivery Vectors
Hydrolytically degradable delivery vectors offer only a limited spatial control of
degradation and release of RNAi molecules. Reductively degradable vectors have
emerged as more suitable alternatives because of the existence of robust and reliable
redox potential gradient that localizes their degradation predominantly to the intra-
cellular space (cytoplasm and nucleus) thus making them more favorable for RNAi
delivery [ 96 ]. Bioreducible delivery vectors with incorporated disulfide bonds into
the vector structure exhibit the capability of responding to the redox potential gra-
dients between the oxidative extracellular environment and the reducing intracel-
lular space. The existence of this redox potential gradient is determined by small
redox molecules like glutathione (GSH) with the assistance of redox enzymes [ 97-
100 ]. The low GSH concentration (1-2 mM) in the plasma allows the nucleic acids
to remain condensed in the delivery vector, while high intracellular GSH level
(>1 mM in the cytoplasm and ~20 mM in the nucleus) [ 101 ] triggers the reduction
of the disulfide bonds, followed by fast disassembly of the polyplexes and release
of the RNAi molecules. Introducing reducible disulfide bonds into the backbone of
the polycations or lipids allows nanoparticles to undergo intracellular thiol-disulfide
exchange reactions, leading to the cleavage of the disulfide bonds and fragmenta-
tion of the polycations or lipids. The increased disassembly rate of the RNAi deliv-
ery vesicles ultimately enhances the knockdown ef fi ciency and decreases cytotoxicity
of the vectors [ 55- 59 ] .
4.4
Future Perspectives
In order to design effective RNAi delivery systems and to advance RNAi therapy
into clinic, countless efforts are being made to improve our understanding of the
intracellular trafficking of the delivery vectors. To design efficient and safe vectors
for RNAi delivery, each and every intracellular barrier including cell uptake, endo-
somal escape, vector disassembly, and, where applicable, nuclear localization
should be taken into consideration. Off-target effects and immune responses should
also be taken into account especially for siRNA delivery. Viral RNAi delivery meth-
ods suffer from several safety and preparation issues and are less feasible for
modifications. On the other hand, synthetic vectors are more versatile in designing,
which allows specific modifications to overcome each barrier. The synthetic deliv-
ery systems have shown great potential, and they promise to not only become useful
research tools but also be applied in future clinical trials.
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