Biomedical Engineering Reference
In-Depth Information
The incorporation of disulphide bridges within the polymer
backbone is an established approach to facilitate intracellular
polyplex disassembly triggered by the cleavage of disulphides by
intracellular reducing agents such as glutathione [51, 52, 64-66].
Electrophoretic migration of siRNA in a polyacrylamide gel shift assay
and morphological changes visualised by atomic force microscopy
(AFM) were used to study particle decomplexation and subsequent
siRNA release after addition of the reducing agent dithiothreitol
(DTT) to mimic intracellular redox conditions. The requirement
for redox conditions for particle disassembly and subsequent
siRNA migration was shown for all reducible polymer systems at
NP 5 and 10. Furthermore redox-induced nanoscale morphological
changes were revealed by AFM. Interestingly, the average size of
the particle increased from ~19 to ~32 nm after addition of DTT,
which suggests a decomplexation process that includes particle
swelling events possibly mediated by electrostatic repulsive forces
between hydrated polymer chains during siRNA liberation. We have
previously observed this phenomenon for redox-activated release of
DNA using a similar polymer [67]. Cellular cytotoxity of polycations
has been shown to be molecular weight dependent attributed to
greater interaction of high charge density with cellular components.
Intracellular breakdown into lower molecular weight fragments is
a strategy to reduce polymer-associated intracellular toxicity [68].
No cytotoxity was observed for the reducible systems containing
excess polymer (NP 20) in two cell lines, suggesting possible redox-
induced polypeptide fragmentation into low molecular polymers
and concomitant reduction in binding affinity to vital intracellular
components.
Intracellular trafficking can be restricted by particle sequestration
within the endosomal-lysosomal compartments as a consequence of
uptake by endocytosis. Endosomal escape is, therefore, crucial and a
prerequisite to enabling siRNA engagement with either cytosolic or
nuclear targets. Movement from the endosomes into the cytoplasm
and nuclear compartments were targeting considerations addressed
with these systems. Live cellular uptake and intracellular trafficking
were investigated in HeLa cells by fluorescent microscopy focused on
particles composed of Alexa488-labelled rCPP-D (NLS 25 : 75 HRP)
and Cy5-labelled siRNA. Fluorophore co-localisation at 2 h post-
transfection suggests incomplete disassembly in a proportion of the
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