Biomedical Engineering Reference
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northern analysis performed using a DNA oligonucleotide probe
complementary to the miRNA-23a guide strand to quantify the
delivery of pre-miRNA. Only the rHB (17%) polymer facilitated
efficient transfection of pre-miRNA. This finding is consistent with
the higher fluorescent uptake and higher knockdown efficiency
observed using this polymer.
Knockdown experiments in H1299 EGFP-expressing cells,
measured by the decrease in mean fluorescence detected by flow
cytometry, was used to find optimal w/w of the range of polymers
at 10, 20, or 40. In all HB polymers, higher w/w induced greater
knockdown with the highest knockdown of ~74% (compared to
non-treated control cells) observed for the rHB 17% w/w ratio 40.
The necessity for excess polymer (high w/w ratio) indicates the
key role of free polymer for particle stability, cellular uptake, and
endosomal escape. The rHB with 25% reducible content showed the
lowest EGFP knockdown of ~25%, which could reflect insufficient
disassembly. Interestingly, the highest reducible content rHB
50% showed an intermediate level of knockdown ~45%, which
suggests that other factors other than reducibility have an effect on
silencing. Furthermore, the polyplexes composed of non-reducible
polymer showed ~53% indicating some degree of disassembly to
allow siRNA interaction in the absence of disulphides. The weak
nature of electrostatic interactions constituting polyplex assembly
may account for this effect. The work does, however, suggest level
and duration of RNAi activity is dependent on the non-reducible/
reducible balance.
The transient nature of synthetic siRNA requires persistent
delivery of the siRNA into the cells for RNAi-based treatment of
chronic diseases. Vector-based viral [34] or plasmid delivery [33]
may overcome this requirement. An alternative approach is the
application of sustained-release technology for controlling the siRNA
release rate. Sustained-release microsphere [77] and nanoparticle
[78] systems based on biodegradable poly(lactide-co-glycolide)
(PLGA) have been developed for the delivery of siRNA. Approaches
based on the degradation properties of PLGA, however, seem most
suited to depot strategies rather than direct entry into cells.
We investigated the possibility to tailor the duration of RNAi
by tuning the intracellular release of the siRNA controlled by
disulphide content. We conducted experiments to investigate the
level of disulphide content on temporal knockdown over a 72 h time
course. The non-reducible polymer showed maximal EGFP silencing
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