Biomedical Engineering Reference
In-Depth Information
The series of rHB polymers increasing in reducible disulphide
content (rHB 17%, rHB 25%, rHB 50%) and control without
disulphide (non-rHB) were used to form polyplexes with conven-
tional siRNA by simple addition and gentle mixing at a weight:
weight (w/w) ratio of 50:1 (polymer:siRNA). AFM studies revealed
the formation of nanoscale systems ~100 nm for all the polymers
(Fig. 6.3). In contrast to the non-reducible system, nanoparticle
incubation with the reducing agent DTT resulted in morphological
changes and accompanying size decreases for all the disulphide-
containing polyplexes; rHB 17% from ~150 to ~33 nm, rHB 25%
from ~110 to ~25 nm, and rHB 50% from ~90 to ~50 nm. Polyplexes
formed with rHB 17% exhibited a greater net positive surface charge
of ~30 mV
compared to 11 mV and 9 mV for rHB 25% and 50%,
respectively, most probably reflecting the higher molecular weight
of this polymer (rHB 17% mw115,000; rHB 25% mw 32,000; rHB
50% mw 40,000) contributing to excess free charges after particle
assembly. Even at w/w 50, all nanoparticle compositions exhibited
non-toxicity in an EGFP-endogenous expressing H1299 cell line after
4 h incubation. This finding is similar to the non-toxicity previously
shown for linear [76] and branched [73] reducible PAMAM polymers.
Interestingly, the non-rHB also showed minimal toxicity in this cell
line, suggesting the non-toxic effects of the hyperbranched PAMAM at
the 50 kDa molecular weight used for this non-reducible polymer.
Cellular uptake studies performed in H1299 cells showed rapid
internalisation for all the polyplexes containing Cy3-labelled siRNA
over the 4 h period investigated; however, greater uptake was
observed for rHB 17%, which could be related to the higher surface
charge facilitating greater interaction with the cellular membrane.
This was accompanied by diffuse fluorescence that indicates siRNA
release. In contrast, punctuate fluorescence was observed for the
non-rHB, rHB 25% and 50 %, which could indicate endosomal
capture or a slower decomplexation rate with these polymers.
The capability of the rHB polymers for delivery of a range of
RNAi triggers other than conventional siRNA was investigated
with cytoplasmic delivery of pre-miRNA that engages upstream of
RISC into DICER. The motivation for its use was driven by reports
of improved silencing for endogenous sequential processing of
miRNA [27], in addition to the potent effects reported for Dicer-
substrate siRNA [17]. H1299 cells were transfected for 4 h with
a series of rHBs (w/w 50) complexed with a 57 nucleotide pre-
miRNA-23a. After 48 h, total RNA was isolated from the cells and
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