Disulfide-Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Article abstract of DOI:10.1002/anie.201900993

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.

Full text of DOI:10.1002/anie.201900993

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Disulfide-unit conjugation enables ultrafast cytosolic
internalization of antisense DNA and siRNA
Authors: Zhaoma Shu, Iku Tanka, Azumi Ota, Daichi Fushihara, Naoko
Abe, Saki Kawaguchi, Kosuke Nakamoto, Fumiaki Tomoike,
Seiichi Tada, Yoshihiro Ito, Yasuaki Kimura, and Hiroshi Abe
This manuscript has been accepted after peer review and appears as an
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using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900993
Angew. Chem. 10.1002/ange.201900993
Link to VoR: http://dx.doi.org/10.1002/anie.201900993
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10.1002/anie.201900993
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COMMUNICATION
Disulfide-unit conjugation enables ultrafast cytosolic
internalization of antisense DNA and siRNA
Zhaoma Shu[a], Iku Tanaka[a], Azumi Ota[a], Daichi Fushihara[a], Naoko Abe[a], Saki Kawaguchi[a],
Kosuke Nakamoto[a], Fumiaki Tomoike[b], Seiichi Tada[c], Yoshihiro Ito[c], Yasuaki Kimura[a] and Hiroshi
Abe*[a][c][d]
Abstract: Development of intracellular delivery methods for
be introduced into cells by making conjugates of CPPs and ONs.
However, since most of the CPPs are cationic, the conjugates
show cytotoxicity.[8] Furthermore, the conjugates can form a
polyion complex and become insoluble.[9] Conjugates of
hydrophobic molecules, such as cholesterol and lipids, and ONs
have also been reported,[10] but because they form micelles,
these conjugates do not exist as single molecules. ON
conjugates with ligands for receptors on the cell surface are also
efficiently taken up by the target cells.[11] For example, it has
been reported that siRNA conjugated with GalNAc (N-acetyl
galactosamine), which is a ligand for the asialoglycoprotein
receptor is efficiently taken up into the primary cultured cells
expressing this receptor.[12] This strategy is useful for targeting
specific cells that express a particular receptor. Finally, most of
the methods described above have problems in endosomal
escape of ONs.[13] In this study, we attempted to develop a
conjugate molecule that universally improves the cellular uptake
of ONs. Based on the background summarized above, we
designed molecules for ON conjugates that satisfy the following
requirements (Fig. 1). (1) Small molecule without charge, (2)
Does not aggregate and exists as a single molecule. (3) Does
not cause cytotoxicity. It has been reported that peptides with
disulfide groups have cell membrane permeability and such a
disulfide unit may fulfill the above requirements. Cyclic disulfide-
modified peptides and small molecules with cell membrane
permeability have also been reported.[14],[15] To our knowledge,
there have been no reports of the use of disulfide unit for the
intracellular delivery of ONs. Therefore, we synthesized series of
ONs with disulfide groups and evaluated their cellular uptake.
Then, we found the modification of repeated linear disulfide unit
with ONs enables the rapid cytosolic internalization of ONs
without the endosomal trap which is triggered by the formation of
a disulfide bond with a thiol group of a protein on the cell surface
and affords potent gene silencing effects (Fig. 1).
antisense DNA and siRNA is important. Previously reported methods
using liposomes or receptor-ligands have
a
problem that
oligonucleotides take several hours or more to reach the cytoplasm
due to long-time residence of oligonucleotides at endosomes. In this
study, we clarified that oligonucleotides modified with low molecular
disulfide units at the terminus reaches the cytoplasm 10 minutes
after the administration to cultured cells. This rapid cytoplasmic
internalization of disulfide-modified oligonucleotides suggests the
existence of an uptake pathway other than endocytosis. In fact, the
mechanistic analysis revealed that the modified oligonucleotides are
efficiently internalized into the cytoplasm through disulfide exchange
reactions with the thiol groups on the cellular surface. Because our
approach solves several critical problems with the currently available
methods for enhancing cellular uptake, including toxicity, undefined
molecular composition, inefficient endosomal escape, and serum
stability, this method may be an effective approach in the medicinal
application of antisense and RNAi methods.
Antisense DNA and siRNA have been widely used for gene
silencing in basic research and in medicinal applications.[1]
Effective delivery of the oligonucleotides (ONs) into the cells is
important for clinical applications.[2] For this reason, delivery
methods for ONs have been extensively studied.[3] A variety of
methods using polymeric materials have been investigated and
[4]
one of the most popular methods utilizes cationic liposomes.
However, the material of the cationic liposomes needs to be 5 to
10 times the weight of the ONs.[5] In addition, the liposome and
the ONs form a complex, and the molecular composition of this
complex cannot be uniformly defined.[6] Therefore, a new
method is required based on
a well-defined molecular
composition that enables intracellular delivery of ONs using low
molecular weight material. One strategy is to make ON
conjugates with small molecules that are membrane permeable.
For example, cell-penetrating peptides (CPPs) are peptide
molecules with a molecular weight equal to or less than that of
ONs. [7] It has been reported that antisense DNA and siRNA can
[a]
Z. Shu., I. Tanaka., A. Ota., D. Fushihara., Dr. N. Abe., S.
Kawaguchi, Dr. K. Nakamoto, Dr. Y. Kimura, Prof. H. Abe,
Chemistry Department, Nagoya University
Furo-cho, Chikusa-Ku, Nagoya, Aichi 464-8602, Japan
E-mail: h-abe@chem.nagoya-u.ac.jp
[b]
[c]
Dr. F. Tomoike
Research Center for Materials Science, Nagoya University
Furo-cho, Chikusa-Ku, Nagoya, Aichi 464-8602, Japan
Dr. S. Tada, Prof. Y. Ito
Emergent Bioengineering Materials Research Team, RIKEN Center
for Emergent Matter Science, 2-1, Hirosawa, Wako-Shi, Saitama,
351-0198, Japan
Figure 1. Conjugation with disulfide units enhanced the permeability of
oligonucleotides.
[d]
Prof. H. Abe
CREST, Japan Science and Technology Agency, Tokyo 102-0076,
Japan
We designed antisense DNA and siRNA molecules having di-
sulfide groups. Because introduction of the disulfide group
within the sequence of the ON strands would impair the
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.201900993
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COMMUNICATION
biological function, we introduced disulfide units at the terminus
of the ON strands by phosphoramidite chemistry. Two types of
phosphoramidite units with a disulfide group were designed (Fig.
2a, Scheme S1). That is, a linear disulfide (LD) unit amidite LDA
having a tertbutyl disulfide group and a cyclic disulfide (CD) unit
amidite CDA having a dithiothreitol (DTT) group. These disulfide
units can be introduced into any position of the ONs with any
number of repeats by an automatic ON synthesizer. In addition,
because they have a phosphate triester structure, there is no
charge on the units.
Antisense DNA (ASD, 1CD, 5CD, 10CD, 1LD, 5LD, and 10LD)
was designed by introducing CD or LD units with 1, 5, or 10
repeats at the 5’ end of 19 nt phosphorothioate antisense DNA
(Fig. 2b). The target sequence of the antisense DNA was the
firefly luciferase gene (961–979 site).[16] These antisense DNAs
were synthesized by a DNA synthesizer and purified using
HPLC and the purity was confirmed by HPLC and MALDI-TOF-
MS (Fig. S1, Table S2). First, the micelle-forming ability of LD-
modified ONs was examined using pyrene, which is an
environment-responsive fluorescent molecule used for
estimating the critical micellar concentration (CMC) (Fig. S3). No
enhancement of fluorescence intensity was observed for 5LD or
10LD at 1 µM and 100 nM. These results suggested that the LD-
modified ONs do not form micelles at 1 µM or less, and exist as
single molecules. Hence, LD-modified ONs may have desirable
properties for clinical applications.
The comparable efficiency of cellular uptake between ASD and
5LD was also confirmed in other cell lines including NIH/3T3 and
A431, which demonstrated the versatility of the disulfide-based
strategy (Fig. S4). The intracellular distribution of 5LD and 5CD
was analyzed by confocal laser scanning microscopy (CLSM).
The image of the ONs at 10 min after lipofection showed that
fluorescent-labeled ASD (ASD*) was co-localized mainly with
the endosome (Fig. 3c), which indicated the endosome trap of
ASD. The images of 5LD and 5CD at the same time point
showed that they were not co-localized with the endosome, but
were quickly distributed in the cytoplasm. This implies that the
disulfide modified ONs internalize into cells via non-endocytotic
pathway. After incubation for 1 h, the uptake of 5LD and 5CD
was increased and good distribution to the cytoplasm was
maintained (Fig. 3d).
Figure 2. a Structure of disulfide phosphoramidites and b sequence of
antisense DNA and siRNA. Underlined parts are phosphorothioate DNA. The
guide strands of siRNA are shown in bold.
Figure 3. a-g; Disulfide unit conjugation enhanced the cellular uptake and
antisense effect. a The cellular uptake of 5LD*, 5CD*, and ASD* measured by
flow cytometry. b Effect of the repeat number of LD on cellular uptake. c The
intracellular distribution of 5LD* and 5CD* compared with ASD* transfected by
Lipofectamine 3000 after 10 min incubation or d after 1 h incubation. e
Inhibitory assays for cellular uptake of 5LD* and 5CD* using DTNB and
iodoacetate. f Gene silencing effect of 5LD and 5CD in dual luciferase assays.
The luminescence signals for each sample were normalized to conditions of
no administration. g Evaluation of cell viability by MTT assays after 24 h
treatment. The experiment was performed three times independently. Data are
presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus
controls; #P < 0.05, ##P < 0.01, ###P < 0.001 versus ASD*/Lipo. MFI is the
abbreviation for mean fluorescence intensity. Sequence names marked with *
means that the ON is fluorescently-labelled.
Next, the cellular uptake was evaluated using the antisense
DNAs labeled with fluorescein (marked with *, Table S1) by flow
cytometric analysis (Fig. 3a). For ONs at concentrations of 1 µM
and 100 nM, the ASD was hardly up-taken without lipofection,
whereas the LD-conjugated DNA 5LD was significantly up-taken.
More efficient uptake was observed as the number of repeats of
the LD unit increased (Fig. 3b). The uptake of 5LD was almost
the same compared with the case using Lipofectamine 3000
(Lipo), and the uptake of 10LD was twice as high. The uptake of
5CD at 1 µM was approximately 70% of that of 5LD (Fig. 3a).
2
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10.1002/anie.201900993
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These results suggested that the designed disulfide unit, not
only promoted cellular uptake, but also enables ultrafast
cytoplasm distribution, which is desirable for clinical use.
endocytosis inhibitors, although the uptake of NR with lipofection
was significantly inhibited (Fig. S6). These inhibition experiments
suggested that 5LR may be directly introduced into the
cytoplasm via disulfide bond formation on the cell surface, which
indicated the mechanism is different from classical endocytosis
internalization. Similarly, to the experiments with antisense DNA,
the gene silencing effect of LD-modified siRNA was evaluated
using HeLa cells stably expressing the luciferase gene (Fig. 4e).
Intracellular uptake experiments were carried out in the
presence of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and
sodium iodoacetate[17] to clarify whether disulfide-modified ONs
are taken up via disulfide bond formation on the cell membrane
surface (Fig. 3e). DTNB inhibited uptake of 5CD and 5LD by 7%
and 11%, respectively. For iodoacetate, the uptake of both 5LD
(82%) and 5CD (55%) was significantly inhibited. It was
suggested that disulfide bond formation may be involved in the
uptake mechanism. Previously, transferrin was suggested to be
a membrane protein involved in the disulfide exchange in the
similar system for peptide delivery.[15] However, it is difficult so
far to mention the responsible membrane protein, since the
unusually efficient cytosolic distribution was observed in our
system, which suggests the different mechanism to that of the
related reported methods. [18]
To evaluate the gene silencing effect, the antisense effects of
5LD and 5CD were investigated by dual luciferase assay using a
HeLa cell line stably expressing firefly and Renilla luciferase
genes. When using ONs at 1 µM or 100 nM concentrations,
lipofection, 5LD, and 5CD showed comparable inhibitory effects
in a concentration-dependent manner (Fig. 3f). In addition, the
cytotoxicity of these intracellular delivery methods was evaluated
by MTT assays. The results indicated that the lipofection method
reduced the cell viability to 90% and 70% at concentrations of 1
and 5 µM, respectively, while 5LD and 5CD did not affect the
cell viability (Fig. 3g).
Next, the LD unit was introduced into siRNA strands and the
cellular uptake and RNAi effects were evaluated. The sequence
of the siRNA used targeted the firefly luciferase gene (867–885
site).[16] The introduction of the LD unit to the 5’ end of the guide
strand was avoided because it is known to be important for
RISC (RNA-induced silencing complex) formation. Several types
of LD-modified siRNAs were designed as follows; 5LR-a: five
repeated LD units were introduced at both 3’ ends of the siRNA
strands, 5LR-b: five repeated LD units were introduced at the 5’
end of the passenger strand and the 3’ end of the guide strand,
10LR-b: 10 repeated LD units were introduced at the 5’ end of
the passenger strand and the 3’ end of the guide strand (Fig. 2b,
Fig. S2). First, the possibility of micelle formation was checked;
fluorescence analysis using pyrene revealed that 5LR-b and
10LR-b were monodisperse and did not form micelles at 1 µM or
less (Fig. S5). Next, the cellular uptake of these modified
siRNAs was evaluated using fluorescently labeled RNAs
(marked with *, Table S1). At concentrations of 1 µM and 100
nM, 10LR-b showed higher intracellular uptake than siRNA
administrated via the lipofection method (Fig. 4a). In addition,
5LR-a and 5LR-b resulted in cell uptake efficiency of
approximately 40% that of lipofection. CLSM imaging analysis
showed that after 10 min, 5LR-b was localized mainly in the
cytoplasm, not in the endosome (Fig. 4b), similar to the results
for the LD-modified DNA 5LD. In contrast, NR transfected by
lipofection was co-localized mainly with the endosome even
after 1 h (Fig. 4c). Next, various inhibitors were tested to
investigate the cellular uptake mechanism. First, the intracellular
uptake of 5LR-b was significantly inhibited by both DTNB and
iodoacetate, which indicated thiols on the cell surface have a
critical role for the internalization of 5LR-b (Fig. 4d). Second, the
intracellular uptake of 5LR-b was not inhibited by three kinds of
Figure 4. a-h Disulfide unit conjugation enhanced the cellular uptake and
knockdown effect of siRNA. a The cellular uptake of LD-conjugated siRNA
measured by flow cytometry. b The intracellular distribution of 5LR-b* and NR*
transfected by Lipofectamine 3000 after 10 min incubation or c after 1 h
incubation. d Inhibitory assays for cellular uptake of 5LR-b* using DTNB and
iodoacetate. e Gene silencing effect of 5LR-a, 5LR-b, and 10LR-b in dual
luciferase assays. The luminescence signals for each sample were normalized
to conditions of no administration. f The real-time qPCR measurement of the
change in ApoB mRNA after 24
h administration of 5LR-ApoB. The
experiment was performed three times independently. Data are presented as
the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus controls; #P <
0.05, ##P < 0.01, ###P < 0.001 versus NR*/Lipo, NR/Lipo or NR-ApoB/Lipo.
MFI is the abbreviation for mean fluorescence intensity. Sequence names
marked with * means that the sequence is fluorescently-labelled. g Knock-
down effect for ApoB by Western-blotting assay. h The quantitative analysis of
ApoB protein expression normalized by β-actin. Western blot experiment was
performed two times independently.
At both concentrations of 1 µM and 100 nM, 5LR-a and 5LR-b
showed a higher silencing effect than ON administrated via
lipofection. Interestingly, 5LR-b, which has LD units on the same
3
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COMMUNICATION
sides of the double-stranded RNA, showed a higher silencing
effect than 5LR-a. Therefore, we concluded that 5LR-b was the
optimal molecular design. The stability of 5LR-b in serum was
evaluated (Figs. 5a, b). After 30 min, the canonical siRNA (NR)
had completely disappeared, whereas 72% of 5LR-b remained.
Since degradation of siRNA in serum is one of major clearance
pathways,[19] this result indicated that LD modification would like
to be beneficial for in vivo applications of siRNA also in terms of
biostability. Compared with classical nanoparticle system (e.g.
liposomes), the stabilization effect might be weaker, but still
many advantages such as efficient cytosolic delivery, low toxicity,
and defined molecular composition would surpass this inferiority.
Scientific Research on Innovative Areas 2707 Middle Molecular
Strategy [18H04398 to H.A] from MEXT, and also by the Otsuka
Toshimi Scholarship Foundation to Z. S. Victoria Muir, PhD,
from Edanz Group (www.edanzediting.com/ac) edited a draft of
this manuscript.
Keywords: cellular uptake • antisense • siRNA • cytosolic
delivery • disulfide
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in our group.
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10.1002/anie.201900993
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Oligo-disulfide Magic!
Z. Shu, I. Tanaka, A. Ota, D. Fushihara,
N. Abe, S. Kawaguchi, K. Nakamoto, F.
Tomoike, S. Tada, Y. Ito, Y. Kimura and
H. Abe *
Disulfide conjugation with antisense
DNA and siRNA enabled efficient and
ultrafast cytosolic uptake of these
bioactive oligonucleotides without
toxicity. The new method represented
herein solved the long-standing
problems of various oligonucleotide
delivery methods and should enhance
therapeutic applications of antisense
DNA and siRNA.
Page No. – Page No.
Disulfide-unit conjugation enables
ultrafast cytosolic internalization of
antisense DNA and siRNA
6
This article is protected by copyright. All rights reserved.