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Fig. 8.7 Protonated structures of PAsp(DET) and PAsp(TEP), according to pH changes in the
cellular microenvironment. Reproduced from [ 80 ] with permission from The Royal Society of
Chemistry
by Kwon has presented stimuli-sensitive chemistry that can be included in the
design of polymeric micelles for siRNA delivery [ 78 ] . Among the structures, acid-
cleavable linkers and acid-protonating groups were identified (Fig. 8.8 ). The syn-
thesis of block copolymers containing stimuli-sensitive groups enables the structure
to (1) undergo conformational changes from a hydrophilic structure to a membrane-
destabilizing form at the low pH in the endosomes or (2) facilitate disassembly of
the nanocarrier resulting in efficient delivery of free siRNA [ 88 ] .
Some of these chemical groups can be linked to a block copolymer to protect the
endosome-disrupting component, converting the charge of (or neutralizing) the
primary amines. That is the case of a system designed by our group, in which the
cationic charges of the dual-functional polycation PEG-PAsp(DET) (the stealth and
endosomal-disrupting functions provided by PEG and PAsp(DET), respectively),
was converted to a net negative charge with the addition of cis -aconitic acid form-
ing PEG-PAsp(DET-Aco) [poly(ethylene glycol)- b -poly{ N -[ N -( N -cis-aconityl-2-
aminoethyl)-2-aminoethyl]aspartamide}]. Namely, charge-conversional polymer
(CCP), the newly formed polyanion comprising cis -aconitylamide enabled an effec-
tive integration of the dual-functional block copolymer to calcium phosphate nano-
particles carrying siRNA through the two carboxylates of the cis -aconityl moiety
[ 53 ]. In the rational hypothesis, the obtained hybrid nanoparticles are stable in pH 7.4,
but at the endosomal pH of 5.5, cleavage of the cis -aconitylamide will take place to
reproduce the endosomal destabilizing polycation PAsp(DET) following to the dis-
assembly of the particles and siRNA release into the cytoplasm (Fig. 8.9 ).
Confocal microscopy revealed a rapid endosomal escape of the labeled siRNA
(Fig. 8.10 ), indicating that the siRNA was released from the nanoparticles in the
cytoplasm and was free to produce the RNAi effect, in agreement with the initial
hypothesis for the design of the system. When the red pixels of cy5-siRNA are co-
localized with the green pixels of lysotracker, the resultant color is yellow, and it is
an indicative that the nanocarrier is still inside the endosomes. When the red pixels
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