Amphiphilic Block Copolymer Based Nanocarriers for Drug and Gene Delivery - Intracellular Delivery: Fundamentals and Applications - page 276

inside the cell in the presence of reducing glutathione abundant in the cytoplasm
but not in blood compartment (Meister and Anderson 1983 ). However, introduction
of thiol groups was found to decreases the electrostatic association sites for the
interaction of PLL and DNA. To deal with this problem, Traut's reagent was used
to introduce the crosslinking thiol groups to the PLL and, meanwhile, avoid the loss
of charge density of the block copolymer segment (Miyata et al. 2004 ).
To promote endosome escape, PEO-PLAA-based block copolymers that con-
tain two or more amino groups in the side chain have been designed to use 'proton
sponge effect' (Kanayama et al. 2006 ; Itaka et al. 2004 ). The primary amine group
at the distal end of the side chain showed a high pKa and was used for complexation
with negatively charged siRNA or DNA. The secondary amine of the side chain
located closer to the polymeric backbone showed a lower pKa and was expected to
provide buffering capacity for proton sponge effect. These block catiomers, were
prepared by the aminolysis of PEO-poly(b-benzyl-L-aspartate) (PEO-PBLA) with
either diethylenetriamine (DET), 4-methyldiethylenetriamine (MDET) or N , N -
diethyldiethylenetriamine (DEDET). Both the PEO-P[Asp(MDET)] and PEO-
P[Asp(DEDET)] polyplex micelles showed an appreciably lower transfection than
the PEO-P[Asp(DET)] polyplex micelles. The optimum transfection efficiency
was obtained by DET substituted PEO-PBLA micelles. In a separate study, dipro-
pylenetriamine (DPT) was reacted with PEO-PBLA. The polyion complex of this
system with siRNA has shown superior transfection efficiency over lipid-based
commercial vector for siRNA delivery i.e., RNAiFect. Free polycations in such
polyplexes substantially contribute to efficient transfection due to proton sponge
effect but also introduce toxic effects.
To reduce the free-polycation mediated toxic effects for in vivo gene delivery,
PEO-PLL based triblock polymers were designed to have multiple functions in the
core. Briefly, the triblock copolymer consisted of PEO, poly((3-morpholinopropyl)
aspartamide) (PMPA) as the low pKa segment with buffering capacity, and PLL as
the high pKa segment to condense DNA (Itaka et al. 2004 ). When plasmid DNA
was encapsulated in PEO-PMPA-PLL it revealed one order of magnitude higher
transfection efficiency than PEO-PLL, which was comparable to the transfection
efficiency of plasmid DNA encapsulated PEI at the corresponding negative to posi-
tive (N/P) ratio, without showing appreciable cytotoxicity. Synthesis of tri-block
copolymers of PEO-PEI-PBLG has also been reported where hydrophobicity of
PBLG was used to induce micellization where PEI was used to incorporate plasmid
DNA effectively (Tian et al. 2005 ).
To achieve active targeting, ligand-modified PEO-PLAA micelles were devel-
oped for gene delivery. For cancer-targeted gene therapy, cyclic RGD peptide
(c(RGDfK)), which specifically recognize integrin avb3 receptors expressed in
cancer and its vascular endothelial cells as well, was conjugated at the end of
PEG-PLL block copolymer (Oba et al. 2008 ). The c(RGDfK)-PEG-PLys/pDNA
polyplex micelle showed remarkably increased transfection efficiency compared
with non-targeted polyplex micelles against HeLa cells which express avb3 integ-
rins. The results from cellular uptake and intracellular distribution study suggested
that c(RGDfK)-modified micelles not only increased cellular uptake but enhanced