Supramolecular nanocarrier of siRNA from PEG-based block catiomer carrying diamine side chain with distinctive pKa directed to enhance intracellular gene silencing

Article abstract of DOI:10.1021/ja047174r

An siRNA nanocarrier formed through self-assembly of PEG-based block catiomer possessing two distinct amino groups with different pK_a values in a side chain was developed. This design provided the carrier with a sufficient siRNA complexation and an assumed buffering capacity in the endosomes, allowing it to exhibit remarkable gene knockdown abilities as well as sufficient serum tolerability. Copyright

Full text of DOI:10.1021/ja047174r

Published on Web 09/30/2004
Supramolecular Nanocarrier of siRNA from PEG-Based Block Catiomer
Carrying Diamine Side Chain with Distinctive pKa Directed To Enhance
Intracellular Gene Silencing
Keiji Itaka,†,‡ Naoki Kanayama,† Nobuhiro Nishiyama,† Woo-Dong Jang,† Yuichi Yamasaki,†
Kozo Nakamura,‡ Hiroshi Kawaguchi,‡ and Kazunori Kataoka*,†
Department of Materials Science and Engineering, Graduate School of Engineering, and Department of
Orthopaedic Surgery, Faculty of Medicine, UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Received May 14, 2004; E-mail: kataoka@bmw.t.u-tokyo.ac.jp
The short double-stranded RNA species, called short interference
RNA (siRNA), can be used to silence the gene expression in a
sequence-specific manner in a process that is known as RNA
interference (RNAi).1 It has become a useful method for the analysis
of gene functions and holds the significant possibility of therapeutic
application. However, to promote an efficient gene knockdown,
especially in an in vivo situation, two substantial issues must be
considered: tolerability under physiological conditions and en-
hanced cellular uptake. Thus, the development of effective siRNA
delivery systems is required.
Recently, a new delivery system of plasmid DNA and oligo-
nucleotides has been developed, based on the micellar assembly
of the poly-ion complex (PIC) of these compounds with block
copolymers consisting of poly(ethylene glycol) (PEG) and poly-
cation segments, leading to the self-assembled structure with a
core-shell architecture (PIC micelles).2 Their excellent properties
for in vivo DNA delivery have been confirmed so far:3 a diameter
around 100 nm with a PEG palisade which enables complexes to
avoid recognition by reticuloendothelial systems, increased nuclease
resistance, increased tolerance under physiological conditions, and
the excellent gene expression in a serum-containing medium.4
We now describe the structural design of a novel block catiomer-
based PIC particularly available for siRNA delivery. PEG-poly-
(3-[(3-aminopropyl)amino]propylaspartamide (PEG-DPT; PEG,
12 000 g/mol, polymerization degree of DPT segment, 68), carrying
a diamine side chain with distinctive pKa, was newly synthesized
by a side-chain aminolysis reaction of PEG-poly(â-benzyl-L-
aspartate) block copolymer (PEG-PBLA) with dipropylene triamine
(DPT) (Figure 1A and Figure S1 in the Supporting Information).
A model compound of a DPT unit, tert-butoxycarbonyl-â-N-3-(3-
aminopropyl)aminopropylamido-R-N-propyl-(L)-aspartamide (Boc-
Asp(DPT)-Pr), was also synthesized (see Supporting Information)
to determine the pKa values of the amino groups.
Boc-Asp(DPT)-Pr clearly gave a two-stage pH-R curve (Figure
1B), from which the pKa values of the primary and secondary amino
groups were determined to be 9.9 and 6.4, respectively. Amino
groups in the PIC of polyamine with polynucleotides including
siRNA generally undergo facilitated protonation due to the zipper
effect or the neighboring group effect during the complexation
process, hampering the proton buffering or the proton sponge
capacity. The unique feature of PEG-DPT is the regulated location
of primary and secondary amino groups in the side chain: the
former, with higher pKa, settles at the distal end of the side chain
to participate in the ion complex formation with phosphate groups
in siRNA molecule, whereas the latter, with lower pKa, located
closer to the polymer backbone, is expected to leave a substantial
Figure 1. (A) Chemical structure of PEG-DPT. (B) Change in protonation
degree (R) with pH for Boc-Asp(DPT)-Pr.
fraction of unprotonated form even in the complex, presumably
due to the lower protonation power and the spatial restriction,
directing to the enhanced intracellular activity of siRNA through
the buffering capacity in the endosomal compartment.
The formation of the siRNA complex with the PEG-DPT was
confirmed by polyacrylamide gel electrophoresis (PAGE) and the
ethidium bromide (EtBr) exclusion assay (see Figure S2 in the
Supporting Information). Note that intercalators such as EtBr bind
the double-stranded (ds) RNA in the same fashion as dsDNA.5 The
free siRNA disappeared at the N/P ratio () [total amines in cationic
segment]/[siRNA phosphates]) >2, in line with a substantial
fluorescence quenching of EtBr at N/P g 2 due to the inaccessibility
of EtBr to the complexed siRNA with PEG-DPT. Furthermore, the
EtBr assay highlights the distinctive role of primary and secondary
amino groups of the side chain in the complex. The PIC of the
double-stranded oligo DNA, composed of sequences similar to the
GL3 targeting siRNA, with PEG-poly(3-dimethylamino)propyl
aspartamide (PEG-DMAPA; pKa ≈ 7.9, see Figure S3 for chemical
structure), revealed a lower degree of EtBr quenching compared
to the PEG-DPT/ds-oligo DNA PIC, even in the region of excess
N/P ratios (see Figure S4), suggesting that the presence of
unprotonated amino groups in the former may hamper the tight
association. PEG-poly(L-lysine) (PEG-PLL; pKa ≈ 9.37, see Figure
S3 for chemical structure) induced EtBr quenching as significantly
as PEG-DPT upon complexation with the ds-oligo DNA, yet the
quenching leveled off at the stoichiometric N/P ratio (N/P ) 1.0)
(Figure S4). This is in sharp contrast with the PEG-DPT/ds-oligo
DNA complex, which showed leveling-off behavior of EtBr
quenching at N/P ≈ 2.0, suggesting that secondary amines with
the lower pKa may be excluded from the ion complexation with
oligonucleotides.
These distinctive features of the PEG-DPT, PEG-DMAPA, and
PEG-PLL complexes indeed correlated with their gene knockdown
abilities. For this evaluation, the GL3 luciferase gene was targeted
† Department of Materials Science and Engineering.
‡ Department of Orthopaedic Surgery.
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10.1021/ja047174r CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. (A) GL3 knockdown by siRNA complexes after serum
co-incubation with serum. (B) Endogenous gene (Lamin A/C) knockdown
after co-incubation (n ) 4; (SD).
of the RNAiFect (Figure 3B). A similar trend was also observed
in HuH-7 cells. However, neither the PEG-PLL nor PEG-DMAPA
system showed any gene knockdown (data not shown). As Lamin
A/C is assumed to abundantly express inside the cells, the threshold
level of the siRNA’s introduction that is necessary to show the
inhibition of gene expression should be significantly higher than
in the case of the luciferase cotransfection study. Thus, these results
of PEG-DPT were very encouraging for the actual therapeutic
knockdown of an endogenous gene by the siRNA delivering
approach.
In conclusion, we reported here an effective siRNA nanocarrier
system based on the self-assembly of the PEG-based block catiomer.
The distinctive polymer design managed both a sufficient siRNA
complexation and a buffering capacity of the endosomes. Notably,
the siRNA/block catiomer complex revealed remarkable knockdown
of the endogenous gene, even after the serum incubation. These
results directed this newly designed system of block catiomer to
have a promising feasibility for in vivo therapeutics.
Figure 2. GL3 luciferase gene knockdown (n ) 4; (SD).
after transfecting two kinds of luciferase pDNAs (pGL3 and pRL;
Promega) to HuH-7 cells. The expression ratio of GL3/RL was
used as the knockdown marker.
Each complex system showed a sufficient knockdown of the GL3
luciferase, while neither the naked siRNA nor the nontargeting
siRNA showed any knockdown (Figure 2). Thus, these results
should be recognized as the veritable RNAi by the GL3-targeting
siRNA delivered into the cytoplasm. Notably, the gene knockdown
abilities of the siRNA/PEG-DPT complex were superior to those
of the other two complexes, especially at higher N/P ratios. At
N/P ) 10, it showed more than an 80% knockdown, which
exceeded the commercial RNAiFect. The cell viability evaluated
by MTT assay was more than 75% of the mock cells, even after
co-incubation with siRNA/PEG-DPT with N/P g 10 (see Figure
S5), suggesting the toxic effect to be eliminated. The siRNA/PEG-
DMAPA complexes showed knockdown abilities to a lesser extent.
Apparently, the loosely associated nature of siRNA, suggested by
the EtBr exclusion assay, is unfavorable for facilitating an effective
intracellular delivery of intact siRNA. PEG-PLL showed a con-
siderable knockdown ability in the low N/P region, yet no particular
enhancement with the increase in the N/P ratios. High efficacy of
PEG-DPT may be characterized by the existence of additional
secondary amines with a lower pKa to promote the internalization
of the siRNA molecules into the cytoplasm through buffering of
the endosomal cavity, as is the case with the polyethylenimine-
based polyplex that shows an enhanced tranfection efficiency at
the higher N/P ratios.6
A serum incubation study was then performed to evaluate the
complex stability under physiological conditions by incubating the
complexes in 50% serum at 37 °C prior to transfection. The siRNA/
PEG-DPT complexes showed comparable abilities of gene knock-
down, even after co-incubation with serum for 30 min (Figure 3A).
In contrast, the lipid-based RNAiFect system was significantly
influenced by the serum incubation, probably due to the nonspecific
association with serum proteins. Thus, these results highlighted the
excellent feasibility of the PEG-DPT/siRNA complex, particularly
under physiological conditions due to the segregation of siRNA
into the PEG microenvironment.
Acknowledgment. This work was financially supported by the
Core Research Program for Evolutional Science and Technology
(CREST) from the Japan Science and Technology Corporation
(JST) as well as by Special Coordination Funds for Promoting
Science and Technology from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT).
Supporting Information Available: Detailed Materials and Meth-
ods section; 1H NMR spectrum of PEG-DPT block copolymer (Figure
S1); results of PAGE and EtBr exclusion assay of PEG-DPT (Figure
S2); chemical structures of PEG-DMAPA and PEG-PLL (Figure S3);
summary of EtBr exclusion assay of these copolymers (Figure S4);
and result of MTT assay (Figure S5). This material is available free of
charge via the Internet at http://pubs.acs.org.
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of 20% of mock samples, which significantly exceeded the ability
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