Biomedical Engineering Reference
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containing histidine-rich peptide (HRP) CKHHHKHHHKC and
SV40 Large T antigen-derived nuclear-localisation-sequence (NLS)
peptide CGAGPKKKRKVC (Fig. 6.1). These polymers were prepared
in the Oupicky laboratory by oxidative copolymerization of cysteine-
capped HRP and NLS peptide subunits to form a linear copolypeptide
backbone covalently linked by reducible disulphide bridges [56]
(Fig. 6.1A). The multiblock strategy aims to install several properties
into a single multifunctional system, namely particle formation,
cellular uptake, cytosolic or nuclear localisation, and siRNA release.
The HPR contains three cationic lysines that facilitate nanoparticle
self-assembly and cellular binding via electrostatic interaction with
phosphate-bearing siRNA and the cellular membrane. HRP contains
histidyl groups that exhibit buffering capacity over the endosomal
and lysosomal pH range and facilitate proton sponge-mediated
endosomal escape and consequent siRNA cytosol localisation
[57, 58]. Targeting to the nuclear compartment is achieved by
inclusion of a SV-40 large T antigen nuclear localisation peptide
sequence [59-61]. Polymer breakdown, subsequent nanoparticle
disassembly, and siRNA release are triggered by the susceptibility of
disulphide linkages to redox-induced cleavage inside the cell [62].
Synthesis of polymers with different molar ratio of HRP and NLS
allows design selection for optimal siRNA delivery to cytoplasmic
or nuclear targets. In a series of experiments, we investigated the
intracellular delivery of nuclear (precursor miRNA transcripts and
transcriptional silencing RNA) or cytoplasmic (21-mer siRNA) RNAi
triggers [54]. Cellular tracking and RNAi activity were investigated
in a range of polymers at different NLS: HRP ratio, rCPP-A (100 : 0),
-B (75 : 25), -C (50 : 50), -D (25 : 75), -E (0 : 100). In order to study
the intracellular effect of the HRP or NLS components, it was crucial
to ensure polyplexes exhibited similar physiochemical properties
such as hydrodynamic diameter and surface charge known to
influence cellular interactions and uptake. Discrete particles in the
size range 200-300 nm were formed at NP 10 with all the polymers
by simple polymer addition to a 21-mer siRNA solution followed
by gentle pipette mixing and incubation for ~1 h. The net positive
surface charge ~20 mV that results from excess polymer is thought
to contribute to cell binding with membrane-associated anionic
proteins, sulphated polysaccarides or proteoglycans [63]. This
allied with nanoscale particles with a predisposition for cellular
endocytosis suggests a non-specific endocytosis process for the
uptake observed by fluorescent microscopy in HeLa cells.
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