Biomedical Engineering Reference
In-Depth Information
Another type of reversible siRNA conjugate was designed by Musacchio et al.
by attaching siRNA to phospholipids (PE) via a disulfide linkage. When inside
the cell, the S-S linkage in such a conjugate would be reduced by intracellular
glutathione (GSH) and liberate the native siRNA into the cytoplasm. To protect
siRNA from nucleolytic degradation en route to the target, the siRNA-S-S-PE
conjugate was incorporated via its hydrophobic PE moiety into stable PEG-
PE-based micelles to form mixed polymeric micellar nanoparticles. In the
obtained mixed siRNA-S-S-PE/PEG-PE micelles, siRNA was well-protected
against degradation by nucleases for at least 24 h, and was released easily
from these nanoparticles in free form in the presence of glutathione (GSH) at a
concentration mimicking intracellular levels. In GFP-C166 endothelial cells,
mixed GFP-siRNA-S-S-PE/PEG-PE micelles downregulated GFP production
50-fold more effectively than free siRNA.
115
Micelles with disulfide crosslinked cores can maintain a stable micellar
structure at physiological ionic strength but are disrupted under reductive
conditions because of the cleavage of disulfide crosslinks, which is desirable for
siRNA release in the reducing intracellular environment. A core-shell-type
polyion complex (PIC) micelle with a disulfide crosslinked core was prepared by
Matsumoto et al. through the assembly of iminothiolane-modified poly(ethylene
glycol)-block-poly(
L
-lysine) [PEG-b-(PLL-IM)] and siRNA at a characteristic
optimum mixing ratio. The PIC micelles showed a spherical shape of y60 nm in
diameter with a narrow distribution, and achieved 100-fold higher siRNA
transfection efficacy compared with non-crosslinked PICs prepared from PEG-
b-poly(
L
-lysine) which were not stable at physiological ionic strength. However,
PICs formed with PEG-b-(PLL-IM) at non-optimal ratios did not assemble into
micellar structures and did not achieve gene silencing following siRNA
transfection.
116
One possible explanation for this observed complexation
behavior between PEG-b-PLL(IM) and siRNA could be related to the
instability of amidines formed with 2-iminothiolane (2-IT). Rearrangement of
2-IT modified amines is known to occur following reactions with amino acids
involving an intramolecular reaction between sulfur and the amidine carbon,
with subsequent release of ammonia and formation of an N-substituted 2-
iminothiolane ring.
117
This five-membered ring structure contains an imine bond
(pK
a
#
6.7), not an amidine bond (pK
a
#
12), thus reducing the positive charge
of the block copolymer at pH 7.4.
118,119
To further investigate the properties of
micellar siRNA delivery vehicles prepared with PEG-b-PLL comprising lysine
amines modified to contain amidine and thiol functionality, lysine modification
was achieved using 2-IT [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,39-
dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)] (Figure 7.10).
Amidines formed with 2-IT were unstable and rearranged into an uncharged
ring structure lacking free thiol functionality, whereas amidines generated with
DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at
higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced
micelles only near stoichiometric molar ratios. In vitro gene silencing was highest
for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to
d
n
4
y
3
n
g
|
8
