Biology Reference
In-Depth Information
by these linkages within the cytoplasm. In this regard, the primary amino groups
of PLL are advantageous once it is possible to introduce thiol groups [
76,
89,
90
] .
A variety of novel PLL derivatives have been synthesized and investigated [
91-
93
]
in an effort to improve the release of nucleic acids by utilizing these linkages for (1)
producing micelles with disulfide cross-linked core for higher stability in the blood-
stream and (2) take advantage of its reducible characteristics for a controlled intra-
cellular siRNA release.
In work from our group (Matsumoto et al.), iminothiolane-modified poly(ethylene
glycol)-
block
-poly( L -lysine) [PEG-
b
-(PLL-IM)] and siRNA were used to prepare
core-shell-type PIC micelles with a disulfide cross-linked core [
93
] . The ~60 nm
micelles maintained the structure at physiological ionic strength but were disrupted
under reductive conditions due to the cleavage of disulfide cross-links. Interestingly,
redox-sensitive PIC micelles presented 100-fold higher siRNA transfection efficacy
compared with non-cross-linked PICs prepared from PEG-
b
-PLL. Furthermore,
deeper analysis of the mechanisms of PIC micelles formation showed that a high
degree of PEG-
b
-PLL modification with 2-iminothiolane resulted in the formation
of
N
-substituted 2-iminothiolane structures in the majority of reacted lysine side
chains [
94
]. Although micelles formed with PEG-
b
-PLL(N2IM-IM) and siRNA
showed high stability in vitro and in the bloodstream, there was a loss of sensitivity
to disulfide reducing conditions which resulted in lower RNAi effect on the cellular
level. Thus, reversible micelle stability is critical to achieve high gene silencing and
a balanced ratio in the reducible disulfide crosslink is necessary.
Acetals and ketals have been included as stimuli-responsive components of
nanocarriers [
78,
95
]. The acid-triggered disruption of the interaction between
siRNA and polymer can be achieved by incorporating the polymers with branches
carrying internal acetal/ketal linkages into the polymeric siRNA micelles [
78
] .
Kwon's group has synthesized linear PEI with grafted primary amines through acid
degradable ketal linkage [
96
]. The siRNA is complexed with the grafted primary
amine of the ketalized PEI to form PIC micelles under physiological pH conditions.
The intracellular release of siRNA is accelerated by the cleavage of the ketal linkage
when the nanocarrier is subjected to acidic conditions. Confocal laser scanning
microscopy observation confirmed the release of siRNA from the ketalized PEI,
probably due to the cleavage of the ketal linkage in the low pH of endosomes/lyso-
somes. Overall, it is important to note that hydrolysis of acetals generates aldehydes
that can react with amines of proteins in the cell and result in toxicity, while hydro-
lysis of ketals will produce ketones.
8.7
Future Perspectives
The effective therapeutic application of RNAi is highly dependent on the development
of nanocarriers for siRNA protection and site-specific delivery. Improved delivery
will lead to increased RNAi efficiency and the decrease in any possible adverse
effects. Successful in vivo delivery requires nanocarriers capable of performing
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