Biomedical Engineering Reference
In-Depth Information
particles, possibly reflecting sequestration within the endosomes
and exposure within a non-reducing environment [69], although
redox enzymes have been suggested to exist within endosomes and
lysosomes [62]. Endosomal compartmentalisation was supported
by co-localisation of a late endosomal plasmid-expressed marker
cyan fluorescent (CFP)-TI-VAMP and directed particle movements
suggestive of endosomal transportation along microtubule motor
proteins [70]. Insufficient endosomal release could be attributed
to insufficient histidyl content (six per peptide) within the polyplex
or requirement for escape times greater than the 2 h investigated.
Fluorescence resolution limits for identification of free siRNA
in contrast to easily distinguishable punctuate fluorescence of
nanoparticles could, however, lead to an underestimation of
intracellular released siRNA. Furthermore, some polyplexes showed
co-localisation with Hoechst-stained nuclei supporting some
degree of endosomal escape and an effect of including an NLS peptide
(25%). NLS-dependent nuclear localisation was demonstrated by
confocal laser scanning microscopy 1 h post-transfection in fixed
HeLa cells (Fig. 6.2). All polyplexes containing an NLS component
showed some accumulation within the nuclei counter-stained
with Hoechst except rCPP-A consisting of 100% NLS possibly
reflecting particle instability or inadequate endosomal escape
due to lack of HRP. In contrast, polyplexes devoid of NLS (rCPP-E)
showed a perinuclear accumulation. These studies demonstrate the
importance for a correct histidine/NLS balance for nanoparticle
stability, endosomal escape, and nuclear trafficking.
The capability for rCPP to facilitate cytoplasmic and nuclear
target engagement and mediate RNAi activity was demonstrated
with conventional siRNA and transcriptional silencing siRNA.
Delivery into cytoplasmic located RISC and consequent silencing with
the rCPP series were evaluated using enhanced green fluorescent
protein (EGFP)-specific siRNA in a H1299 endogenous-expressing
EGFP cell line. Gradual increase in gene silencing, revealed by
decreases in EGFP mean fluorescence intensity detected by flow
cytometry, was observed from polymer A to E that correlates with
increased HPR content. Nanoparticle formed at both NP 5 and 10
followed this trend with greatest gene silencing ~50% 48 h post-
transfection shown for polymer E containing 100% HPR. The 50%
gene silencing level may be due to insufficient endosomal escape;
however, silencing indicates a degree of cytoplasmic localisation.
Search WWH ::
Custom Search