Angewandte
Chemie
DOI: 10.1002/anie.201300178
siRNA Conjugates
Acidic pH-Responsive siRNA Conjugate for Reversible Carrier
Stability and Accelerated Endosomal Escape with Reduced IFNa-
Associated Immune Response**
Hiroyasu Takemoto, Kanjiro Miyata,* Shota Hattori, Takehiko Ishii, Tomoya Suma,
Satoshi Uchida, Nobuhiro Nishiyama, and Kazunori Kataoka*
Small interfering RNA (siRNA) has garnered much interest
as a potential drug because of its strong gene-silencing
activity.[1] Toward the success in siRNA therapeutics, many
strategies have been developed for efficient siRNA delivery
into the cytosol of target cells.[2] Among them, siRNA
conjugates have arisen as one of the promising strategies in
siRNA delivery, as siRNA can be readily conjugated to
a functional molecule to acquire the ability of “programmed
transfer” to the target sites.[3] Indeed, several ligand mole-
cules, such as lactose and RGD peptide, were conjugated with
siRNA for site- (or cell)-specific delivery.[3] Furthermore,
multimolecular siRNA conjugates enable stable polyion
complex (PIC) formation because of the increased electro-
static interactions with polycations, leading to facilitated
cellular uptake through charge neutralization of siRNA and
also protection of siRNA from enzymatic degradations.[4]
However, those siRNA conjugates potentially stimulate
immune responses through the activation of toll-like recep-
tor 3 and/or protein kinase R,[4,5] and thus they are desired to
disintegrate into monomeric siRNAs (mono-siRNAs) in the
cell for reduced immune responses.[4] Meanwhile, considering
that macromolecular drugs, including siRNA and its conju-
gates, would be taken up by cells through endocytosis and
then delivered to the late endosome toward lysosomal
degradation, siRNA needs to escape from the endosome
into the cytosol for efficient gene silencing.[6] Therefore,
design of a smart siRNA conjugate for programmed endo-
somal escape and release of mono-siRNA is a great challenge
for successful siRNA delivery.
Herein, we developed a smart siRNA conjugate to fulfill
the multifunctionality desired for enhanced siRNA delivery
with reduced immunogenicity; that is, reversible PIC stability,
endosomal escapability, and mono-siRNA releasability, based
on a single chemical process. It is known that maleic acid
amide (MAA) is relatively stable at extracellular neutral pH,
while rapidly hydrolyzed at endosomal acidic pH.[7] Thus, we
utilized this MAA chemistry as an acid-labile anionic moiety
for linking siRNA to an endosome-disrupting polycation and
concurrently converting the cationic sites into a biologically
inert anionic derivative.[8] In design, the MAA-based con-
jugate is expected to improve the PIC stability through
increased electrostatic interaction, while degrading the MAA
moieties in the endosome for triggering three actions:
1) complex destabilization through unbalanced charges
within PICs; 2) endosome disruption with the regenerated
parent polycation; and 3) mono-siRNA release by MAA
cleavage (Figure 1a). Figure 1b shows the chemical structure
of siRNA-releasable/endosome-disrupting conjugate (REC),
in which several siRNA molecules are grafted into the
endosome-disrupting polymer side chains by the MAA
linkage. The parent polycation is a polyaspartamide deriva-
tive with two repeating units of aminoethylene in each side
chain (termed PAsp(DET)), which destabilizes the endo-
somal membrane integrity with the cationic diprotonated side
chains to accelerate endosomal escape of the payload.[9]
A precursor polyanion was synthesized from PAsp(DET)
to have a dibenzyl cyclooctyne (DBCO) group by MAA
linkage as a conjugation site for siRNA. Then, an azide-
modified siRNA (azide-siRNA) was reacted with the DBCO
group in the polyanion side chains. Notably, the size exclusion
chromatography (Supporting Information, Figure S5) con-
firmed that more than 95% of azide-siRNAs were conjugated
to the polymer backbone utilizing a freeze–thaw treatment
for the generation of a highly concentrated reactant phase.[10]
This successful conjugation at the quite high rate allows the
use of the obtained conjugate without further purification. As
a result, about 30% of DBCO groups in the polymer side
chains reacted with azide-siRNA; that is, about 5 siRNAs
contained in the conjugate (Figure 1b). To investigate the
[*] H. Takemoto, Dr. K. Kataoka
Department of Materials Engineering, The University of Tokyo
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 (Japan)
E-mail: kataoka@bmw.t.u-tokyo.ac.jp
S. Hattori, Dr. T. Ishii, T. Suma
Department of Bioengineering, The University of Tokyo
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 (Japan)
Dr. K. Miyata, S. Uchida
Division of Clinical Biotechnology, Center for Disease Biology and
Integrative Medicine, The University of Tokyo
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan)
E-mail: miyata@bmw.t.u-tokyo.ac.jp
Dr. N. Nishiyama
Polymer Chemistry Division, Chemical Resources Laboratory
Tokyo Institute of Technology, R1-11
4259 Nagatsuta, Midori-ku, Yokohama 226-8503 (Japan)
[**] This research is supported by the Japan Society for the Promotion of
Science (JSPS) through the “Funding Program for World-Leading
Innovative R&D on Science and Technology (FIRST Program),” and
Health and Labour Sciences Research Grants Research on Medical
Device Development, Ministry of Health, Labour and Welfare. H.T.
thanks the Research Fellowships of the Japan Society for the
Promotion of Science for Young Scientists (JSPS).
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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