A smart DNAzyme-MnO2 nanosystem for efficient gene silencing

Article abstract of DOI:10.1002/anie.201411417

DNAzymes hold promise for gene-silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme-MnO₂ nanosystem for gene-silencing therapy. MnO₂ nanosheets adsorb chlorin e6-labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit ¹O₂ generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO₂ is reduced to Mn²⁺ ions, which serve as cofactors of 10-23 DNAzyme for gene silencing. The release of Ce6 generates ¹O₂ for more efficient photodynamic therapy. The Mn²⁺ ions also enhance magnetic resonance contrast, providing GSH-activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.

Full text of DOI:10.1002/anie.201411417

Angewandte
Chemie
DOI: 10.1002/anie.201411417
Cancer Therapy
A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing**
Huanhuan Fan, Zilong Zhao, Guobei Yan, Xiaobing Zhang, Chao Yang, Hongmin Meng,
Zhuo Chen, Hui Liu, and Weihong Tan*
Dedicated to Professor Ru-Qin Yu on the occasion of his 80th birthday
Abstract: DNAzymes hold promise for gene-silencing therapy,
but the lack of sufficient cofactors in the cell cytoplasm, poor
membrane permeability, and poor biostability have limited the
use of DNAzymes in therapeutics. We report a DNAzyme–
MnO2 nanosystem for gene-silencing therapy. MnO2 nano-
sheets adsorb chlorin e6-labelled DNAzymes (Ce6), protect
them from enzymatic digestion, and efficiently deliver them
tive gene silencing, few reports have addressed the potential
application of RNA-cleaving DNAzymes as therapeutic gene-
silencing agents,[7] the main barrier being the need for a high
cofactor concentration to maintain catalytic activity. For
example, the most widely investigated DNAzyme for RNA
cleavage is 10–23 DNAzyme,[8] which requires at least 5 mm
of Mg2+ ions to form the catalytic domain and efficiently
cleave the substrate. However, with the concentration of free
Mg2+ ions in animal cells ranging from 0.2 to 2 mm,[9] the
formation of the 10–23 DNAzyme catalytic domain cannot be
facilitated, thus resulting in low intracellular catalytic activity.
Its poor cell penetration and equally poor stability in biofluids
also limit the application of 10–23 DNAzyme in gene silenc-
ing.[10] Therefore, the development of a smart carrier that can
enhance the cellular uptake of DNAzymes, protect DNA-
zymes from endogenous nuclease digestion, and self-generate
cofactors in situ in the cytoplasm for catalysis is highly
desired.
1
into cells. The nanosystem can also inhibit O2 generation by
Ce6 in the circulatory system. In the presence of intracellular
glutathione (GSH), MnO2 is reduced to Mn2+ ions, which serve
as cofactors of 10–23 DNAzyme for gene silencing. The release
of Ce6 generates 1O2 for more efficient photodynamic therapy.
The Mn2+ ions also enhance magnetic resonance contrast,
providing GSH-activated magnetic resonance imaging (MRI)
of tumor cells. The integration of fluorescence recovery and
MRI activation provides fluorescence/MRI bimodality for
monitoring the delivery of DNAzymes.
D
NAzymes, generated through in vitro selection processes,
Ultrathin MnO2 nanosheets, typical electrode materials
for energy storage, have attracted extensive attention in
bioanalysis, cell imaging, and drug delivery as a result of their
appealing physicochemical properties.[11] First, MnO2 nano-
sheets can strongly adsorb ssDNA by physisorption between
nucleobases and MnO2 nanosheets, which can facilitate the
endocytosis of ssDNA. Second, MnO2 nanosheets have an
intense and broad optical absorption spectrum (l ꢀ 200–
600 nm), making them an efficient broad-spectrum fluores-
cence quencher. Third, MnO2 nanosheets can be reduced to
Mn2+ ions by intracellular glutathione (GSH). The produced
Mn2+ ions can then be used as efficient cofactors of 10–
23 DNAzyme for gene silencing.[12] Meanwhile, the reduction
of MnO2 can also provide activatable magnetic resonance and
fluorescence signaling to monitor the efficacy of delivery.
Therefore, MnO2 nanosheets provide a potent nanocarrier for
delivering DNAzyme into cells for gene silencing and
simultaneous monitoring of delivery efficacy through cellular
imaging.
In this work, a DNAzyme–MnO2 nanosystem was pre-
pared by the physisorption of nucleobases on MnO2 nano-
sheets for efficient gene-silencing therapy, as shown in
Scheme 1. In this design, Ce6-labelled DNAzyme acts as an
agent for gene silencing, photodynamic therapy, and fluores-
cence imaging. MnO2 nanosheets have multiple roles, serving
as the nanocarrier for the DNAzyme, as a potential provider
of cofactor (Mn2+) for 10–23 DNAzyme, as a quencher for
singlet oxygen generation and fluorescence from Ce6, and as
an activatable MRI contrast agent. In the Ce6–DNAzyme–
MnO2 nanosystem, DNAzyme is protected from enzymatic
are single-stranded DNA (ssDNA) catalysts that can catalyze
a wide variety of reactions, such as RNA or DNA cleavage
and ligation or DNA phosphorylation.[1] Based on specific
cofactor dependence and potent catalytic ability, especially
nucleic acid cleavage,[2] DNAzymes have been extensively
used to develop highly sensitive and specific sensing platforms
for metal ions, small molecules, and biomacromolecules by
integrating various signal transduction mechanisms, such as
fluorescence,[3] colorimetry,[4] electrochemistry,[5] and electro-
chemiluminescence.[6] However, in spite of their multiple
strong enzymatic turnover properties and promise for selec-
[*] H. Fan,[+] Dr. Z. Zhao,[+] G. Yan, Prof. X. Zhang, Dr. C. Yang, H. Meng,
Z. Chen, H. Liu, Prof. W. Tan
Molecular Science and Biomedicine Laboratory
State Key Laboratory of Chemo/Bio-Sensing and Chemometrics
College of Chemistry and Chemical Engineering, College of Biology
Collaborative Innovation Center for Chemistry and Molecular
Medicine, Hunan University, Changsha, 410082 (China)
E-mail: tan@chem.ufl.edu
[+] These authors contributed equally to this work.
[**] This work was supported by the National Key Scientific Program of
China (2011CB911000), the National Key Basic Research Program
of China (No. 2013CB932702), the NSFC (Grants 21325520,
21327009, 21405041, J1210040, 21177036), the Foundation for
Innovative Research Groups of NSFC (Grant 21221003), the
National Instrumentation Program (2011YQ030124), and the
Hunan Provincial Natural Science Foundation (Grant 11JJ1002).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201411417.
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Figure 1. Agarose gel electrophoresis analysis of DNAzyme catalytic
efficiency with a) Mn2+ and b) Mg2+ as the cofactors. The substrate
and DNAzyme concentrations were both 2.5 mm. Cleavage experiments
were carried out at 378C for 60 min.
Scheme 1. Activated mechanism of the Ce6–DNAzyme–MnO2 nano-
system for gene silencing and PDT. The nanosheet can adsorb and
efficiently deliver DNAzymes into cells and protect them from enzy-
matic digestion. In the presence of cellular GSH, the MnO2 nanosheet
is reduced to Mn2+ ions, and DNAzyme is freed for efficient gene
silencing with in situ generated Mn2+ as the cofactor. Ce6 is also
released to generate singlet oxygen for PDT after irradiation.
cleave the substrate within 60 min. However, under the
same conditions, a concentration of 15 mm was necessary for
Mg2+ ions, indicating that the Mn2+ ion was the more efficient
cofactor. The effect of incubation time on the efficiency of
RNA cleavage of 10–23 DNAzyme was further investigated
by agar electrophoresis and fluorescence analysis. It was
found that the effect of Mn2+ ions on the cleavage efficiency
was proportional to incubation time and that 2.5 mm of
substrate mRNA could be cleaved completely by 2.5 mm of
DNAzyme in the presence of 500 mm of Mn2+ ions at 378C
within 60 min (Figure S3a and S3b). These results demon-
strate that the MnO2 nanosheet can be an efficient pro-
cofactor for 10–23 DNAzyme for gene silencing.
The excellent fluorescence quenching ability of MnO2
nanosheets made it possible to test the amount of
10–23 DNAzyme adsorbed on the MnO2 nanosheets by
fluorescence analysis. Accordingly, when 100 nm of carboxy-
fluorescein-labelled DNAzyme (FAM–DNAzyme) was
mixed with different concentrations of MnO2 nanosheets, it
was found that the fluorescence of FAM was gradually
decreased with the increasing concentration of introduced
nanosheets, until it was totally quenched at a concentration of
26. 81 mgmLÀ1 (Figure S4). At this concentration, the amount
of adsorbed DNAzyme on 1 mg of the as-prepared MnO2
nanosheets was calculated to be about 3.26 pmol.
The instability of DNAzymes in biofluids has been
a factor limiting their clinical application. To identify whether
the noncovalent adsorption of DNAzyme onto MnO2 nano-
sheets could defend against enzymatic digestion, the fluores-
cence change of free FAM-labelled ssDNA and ssDNA–
MnO2 nanocomplexes, respectively, in the presence of
deoxyribonuclease I (DNase I) was investigated. It can be
seen in Figure S5 that the free molecular beacon (MB),
specifically FAM, was gradually cleaved by DNase I.
However, even when DNAzyme–MnO2 or MB–MnO2 nano-
complexes were treated with 1 UmLÀ1 DNase I (significantly
greater than what would be found in the cellular environ-
ment) for one hour, no obvious fluorescence changes were
detected. These experimental results demonstrate that
DNAzyme can be protected from DNase I cleavage after
adsorption onto the MnO2 nanosheets.
digestion and can be efficiently delivered into the cytoplasm.
Once endocytosed, the nanosystem is decomposed by the
reduction of MnO2 nanosheets by intracellular GSH, gener-
ating
a
large amount of Mn2+ ions as cofactors for
10–23 DNAzyme cleavage to target RNA. The therapeutic
efficacy can be further enhanced by the photodynamic effect
of Ce6. Meanwhile, fluorescence recovery and activation of
an MRI contrast agent, accompanied by the dissolution of
MnO2 nanosheets, can provide a fluorescence/MRI bimodal
signal for monitoring the efficacy of delivery.
To construct the DNAzyme–MnO2 nanosystem, MnO2
nanosheets were prepared by ultrasonicating bulk MnO2,
which was synthesized using H2O2 to oxidize MnCl2 in the
presence of tetramethylammonium hydroxide.[13] The product
was identified by X-ray photoelectron spectroscopy (see
Figure S1 in the Supporting Information). The results from
transmission electron microscopy (TEM) and atomic force
microscopy (AFM) indicated that the as-prepared MnO2 has
a sheet-like structure about 1.5 nm in thickness (Figure S2).
The as-prepared MnO2 had an intense UV/Vis absorption
with a band centered at l = 360 nm in the absorption
spectrum. Based on the high efficacy and the potential
flexibility in the design of recognition arms,[8b,14]
10–23 DNAzyme (Table S1) was chosen as a model DNA-
zyme to cleave the human early growth response-1 (EGR-1)
gene, which has been reported to regulate breast cancer cell
proliferation, migration, chemoinvasion, and xenograft
growth in nude mice.[15]
To investigate whether the produced Mn2+ ions from the
reduction of MnO2 nanosheets could act as cofactors for
10–23 DNAzyme, the effect of Mn2+ on the catalytic activity
of DNAzyme for RNA cleavage was analyzed by agar
electrophoresis. It can be seen in Figure 1 that 0.2 mm of
Mn2+ ions could trigger 10–23 DNAzyme to completely
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Reduced substance-activated fluorescence enhancement
and singlet oxygen generation from Ce6 were then inves-
tigated by fluorescence analysis and using the reagent singlet
oxygen sensor green (SOSG), respectively. As shown in
Figure S6, the Ce6–DNAzyme–MnO2 solution presented
a low fluorescence signal after irradiation with light at l =
404 nm. However, an 8.1-fold and 22-fold enhancement of
Ce6 fluorescence and a 2.8-fold and 5.2-fold enhancement of
SOSG fluorescence was detected in the presence of 0.25 mm
and 0.5 mm of dl-dithiothreitol (DTT), respectively. These
results indicate that MnO2 nanosheets can efficiently quench
fluorescence and inhibit singlet oxygen generation from Ce6
to decrease phototoxicity during circulation. Additionally, the
results show that the introduction of a reduced substance (e.g.
GSH) can trigger the disintegration of MnO2 nanosheets, in
turn causing the recovery of fluorescence and singlet oxygen
generation of Ce6 for cellular imaging and photodynamic
therapy (PDT).
that the MnO2 nanosheets can efficiently deliver Ce6-labelled
DNAzymes into cells for therapeutic applications.
The intracellular manganese contents in MCF-7 cells after
treatment with MnO2 nanosheets was also analyzed by
inductively coupled plasma optical emission spectrometry
(ICP–OES). The manganese concentrations in MCF-7 cells
incubated with 21.8 mgmLÀ1 and 43.6 mgmLÀ1 MnO2 nano-
sheets were 0.08 pg/cell and 0.21 pg/cell, respectively (Fig-
ure S8). Given the small volume of a single cell (the average
diameter of a MCF-7 cell detached by trypsin is about 25 mm),
MnO2 nanosheets can provide
a sufficient number of
Mn2+ ions to activate 10–23 DNAzyme after reduction. Com-
pared with MnO2 nanosheets, the Mn2+ product also has
a higher longitudinal relaxivity r1 and transverse relaxivity r2
than MnO2 nanosheets (Figure 3a,c and b,d). After treat-
Next, the efficacy of the delivery of MnO2 nanosheets
was examined, comparing the internalization of the
Ce6–DNAzyme–MnO2 nanosystem and that of free DNA-
zyme in MCF-7 breast-cancer cells. As shown in confocal laser
scanning microscopy analysis (Figure 2), MCF-7 cells treated
Figure 3. Plots of a) r1 and b) r2 versus [Mn] for a MnO2 nanosheet
!
&
solution ( ) and a MnO2 nanosheet solution treated with GSH ( ).
c) T1-weighted and d) T2-weighted MRI results obtained from (a) and
(b). Left to right: [Mn]=0, 0.2, 0.4, 0.8, and 1.6 mm. The top and
bottom rows in (c) and (d) correspond to MnO2 nanosheets in the
absence and presence of GSH, respectively. e) T1-weighted (upper)
and T2-weighted (lower) images of MCF-7 cells treated with MnO2
nanosheets at concentrations of 0, 21.8, and 43.6 mgmLÀ1
.
ment with the nanosystem, MCF-7 cells presented enhanced
magnetic resonance contrast (Figure 3e), providing GSH-
activated MRI for tumor-cell imaging. Thus, the integration of
fluorescence recovery and MRI activation, accompanied and
promoted by the disintegration of MnO2 nanosheets, provides
fluorescence/MRI bimodality for monitoring the delivery
efficacy of DNAzymes.
Figure 2. Confocal fluorescence images of MCF-7 cells without treat-
ment (upper) and with treatment with Ce6–DNAzyme (middle) and
the Ce6–DNAzyme–MnO2 nanosystem (lower). The total cellular DNA
was stained with DAPI. In all images scale bars=50 mm. Merge=
overlay of Ce6 and DAPI channels. DIC=differential interference
contrast image.
To identify whether EGR-1 mRNA was really cleaved by
the nanosystem, we first analyzed the expression of
EGR-1 mRNA in MCF-7 cells treated with or without the
DNAzyme–MnO2 nanosystem by quantitative real-time PCR
analysis. As shown in Figure 4a, mRNA level of EGR-1 was
inhibited by 60% after treatment with the nanosystem
containing 5 mm of DNAzyme (DNAzyme–MnO2). However,
equivalent quantities of free DNAzyme, the inactive control
DNA, and the control DNA–MnO2 nanocomplex had little
effect on the expression of EGR-1 mRNA. The inhibitory
effect of the nanosystem on EGR-1 protein expression was
further studied. It could be clearly seen in western blot
analysis that EGR-1 protein expression decreased in cells
with the Ce6–DNAzyme–MnO2 nanosystem presented stron-
ger fluorescence signals in the cytoplasm than those treated
with free Ce6–DNAzyme, indicating that the MnO2 nano-
sheets could enhance the cellular uptake of DNAzyme.
Internalization was also investigated by using flow cytometry.
After the fluorescence signal on the cell surface was removed,
cells incubated with the Ce6–DNAzyme–MnO2 nanosystem
showed significantly higher fluorescence intensity than those
treated with free Ce6–DNAzyme, in good agreement with the
results of confocal microscopy (Figure S7). These data suggest
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was further identified using a soft-agar
colony assay, in which the number and
volume of cell colonies treated with nano-
system were clearly fewer than those of cell
colonies treated with free DNAzyme. Addi-
tionally, only 18% of antiproliferation was
detected when MCF-7 cells were treated
with DNAzyme–graphene oxide (DNA-
zyme–GO) nanocomplexes containing 5 mm
of DNAzyme (Figure S10), which was
slightly higher than the antiproliferation
efficacy of free DNAzyme and much lower
than that of DNAzyme–MnO2 nanocom-
plexes. The difference in antiproliferation
efficacy between DNAzyme–MnO2 and
DNAzyme–GO could be because a large
amount of Mn2+ ions is generated after
DNAzyme–MnO2 is internalized into cells
to act as efficient cofactors to facilitate 10–
23 DNAzyme RNA cleavage. These results
show that the MnO2 nanosheet is both
a potent delivery tool and an efficient
cofactor provider to facilitate DNAzyme-
based gene-silencing therapy.
Figure 4. a) Reverse transcriptase quantitative PCR analysis of EGR-1 expression in MCF-7
cells treated with 5 mm control DNA, the control DNA–MnO2 nanosystem, 5 mm DNAzyme,
and the DNAzyme–MnO2 nanosystem. b) Western blot analysis with antibodies against
EGR-1 and b-actin. Samples were treated with the systems used in (a). c) Immunofluor-
escence analysis with antibodies against EGR-1. MCF-7 cells were treated with the same
systems as in (a). Samples from top to bottom: untreated cells, cells treated with control
DNA, the control DNA–MnO2, the DNAzyme, and with the DNAzyme–MnO2 nanosystem.
Merge=overlay of EGR-1 and DAPI channels. Concentration of MnO2 nanosheets=
40.2 mgmLÀ1. Scale bars in all images in (c)=50mm.
Based on the fact that DNA is easy to
synthesize and modify with other therapeu-
tic agents, photosensitizer Ce6 was intro-
duced to enhance the therapeutic efficacy of
the DNAzyme–MnO2 nanosystem. The syn-
ergistic therapeutic efficacy of DNAzyme-
based gene silencing and PDT of the Ce6–
DNAzyme–MnO2 nanosystem was further
treated with the DNAzyme–MnO2 nanosystem in contrast to
cells treated with free DNAzyme, the control DNA, and the
control DNA–MnO2 nanocomplex (Figure 4b). The inhibi-
tory effect of the nanosystem on protein expression was also
identified by immunofluorescence assays. The relatively small
knockdown in EGR-1 detected in the immunofluorescence
results may have to do with the specificity of the antibody in
our experiments. As shown in Figure 4c, cells treated with the
DNAzyme–MnO2 nanosystem exhibited weaker fluorescence
signals in the nucleus than those treated with free DNAzyme,
the control DNA, and the control DNA–MnO2 nanocomplex.
The results demonstrated strongly that the DNAzyme–MnO2
nanosystem could efficiently cleave its target mRNA and
decrease the expression of the translated product of the target
mRNA, providing a powerful gene-silencing tool.
investigated by using an MTS assay. As shown in Figure 6,
under the same conditions the DNAzyme–MnO2 nanosystem
and the Ce6-control DNA–MnO2 nanocomplex inhibited the
proliferation of MCF-7 cells by 35% and 54%, respectively,
while the Ce6–DNAzyme–MnO2 nanosystem inhibited cell
proliferation by 78%, demonstrating a remarkably improved
and synergistic therapeutic effect. In contrast, an equivalent
To investigate the gene silencing therapeutic efficacy of
the DNAzyme–MnO2 nanosystem, the proliferation of MCF-
7 cells treated with a nanosystem containing different
concentrations of DNAzymes was evaluated using an MTS
assay and a soft-agar colony assay (MTS = 3-(4,5-dimethyl-
thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium). As shown in Figure 5a, cell proliferation
gradually decreased with increasing concentration of DNA-
zyme. A nanosystem containing 5 mm of DNAzyme could
induce 73% of cell antiproliferation. However, free DNA-
zyme at 5 mm caused only 15% of cell antiproliferation. The
potent antiproliferation of the nanosystem on MCF-7 cells
Figure 5. a) Gene-silencing therapy with the DNAzyme–MnO2 nano-
system, in which MCF-7 cells were treated with different concentra-
tions of DNAzyme and the DNAzyme–MnO2 nanosystem. b) Statistical
analysis of results from the soft-agar colony assay. MCF-7 cells were
treated with MnO2 nanosheets, 2.5 mm DNAzyme, and the DNAzyme–
MnO2 nanosystem at [DNAzyme]=2.5 mm. P values were calculated by
t-test. ** P<0.01. c) Confocal images of samples from (b). The
concentration of MnO2 nanosheets was 40.2 mgmLÀ1
.
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Received: November 25, 2014
Revised: January 23, 2015
Published online: && &&, &&&&
Keywords: DNAzyme · gene silencing · manganese ·
.
nanotechnology · photodynamic therapy
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

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Angewandte
Communications
Communications
Cancer Therapy
Multitasking: A smart carrier for DNA-
zymes has been developed in which
MnO2 nanosheets are able to enhance
cellular uptake of DNAzymes, protect
them from endogenous nucleasediges-
tion, and self-generate in situ cofactors
(Mn2+ ions) in the cell cytoplasm to
maintain the catalytic activity of 10–
23 DNAzyme for RNA cleavage and gene
silencing. Ce6–DNAzyme=chlorin e6-la-
belled DNAzyme.
H. Fan, Z. Zhao, G. Yan, X. Zhang,
C. Yang, H. Meng, Z. Chen, H. Liu,
W. Tan*
&&&& — &&&&
A Smart DNAzyme–MnO2 Nanosystem
for Efficient Gene Silencing
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!