Biomedical Engineering Reference
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polymer A composed of 100% NLS compared to minimal reduction
(~10%) with polymer E without NLS. The capability for slight
reductions without NLS could reflect cellular division during the
48 h period. The dependency of NLS inclusion for nuclear silencing
indicates retained polymer association to the siRNA during the
nuclear trafficking step. This suggests incomplete redox-mediated
disassembly of the nanoparticle. High glutathione levels in the
cytosol have been reported [62, 69]; however, nuclear-localised
glutathione has been found [71]. Incomplete removal of the polymer
may be exploited to ensure polymer-mediated nuclear trafficking
prior to complete disassembly within the nucleus, enabling nuclear
target interactions. It would be interesting to determine the amount
retained or possible adverse effects on target engagement due to
steric hindrance. This is an underexplored field; however, we have
previously used restriction-site cleavage in plasmid DNA as a method
to investigate the availability to specific interactions before and after
redox-induced removal of polymer material [67].
Downregulation of miRNA associated with particular disease
states and the potent RNAi effects observed with nuclear-processed
primary miRNA transcripts (pri-miRNA) are the motivation for pri-
miRNA delivery to the nuclear compartment. We investigated nuclear
delivery and subsequent nuclear processing of pri-miRNA23a
(183 nt) into mature miRNA23a (21 nt) effector molecules in the
cytoplasm by inclusion of a NLS peptide derived from the importin
α
binding SV40 large T antigen. The RNA was isolated 48 h post-
transfection in HeLa cells and the levels of mature RNA identified
by northern analysis using an oligonucleotide probe complementary
to miRNA23a. We found there was an NLS-dependent effect on the
amount of mature miRNA detected. Increased levels of detected
mature miRNA followed increases in the NLS content from rCPP-E
(0% NLS) to rCPP-D (25% NLS) to rCPP-C (50% NLS), seemingly
reflecting improved trafficking to the nucleus required for entry
into the miRNA biogenesis pathway. Interestingly, further increase
in NLS to 75% (rCPP-B) and 100% (rCPP-A) resulted in a decline
in the amount of miRNA detected. This may reflect insufficient
histidine content in these polymers, which limits endosomal release
required for nuclear engagement. The low histidine content and
concomitant reduced cationic lysines may also result in polyplex
instability and consequent removal of the NLS polymer prior to
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