Simultaneous Imaging of Endogenous Survivin mRNA and On-Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher

Article abstract of DOI:10.1002/smll.201700569

The design of multifunctional drug delivery systems capable of simultaneous target detection, imaging, and therapeutics in live mammalian cells is critical for biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs) chemically modified with a small-molecule dark quencher, followed by sequential drug encapsulation, MSN capping with a dye-labeled antisense oligonucleotide, and bioorthogonal surface modification with cell-penetrating poly(disulfide)s, the authors have successfully developed the first mesoporous silica nanoquencher (qMSN), characterized by high drug-loading and endocytosis-independent cell uptake, which is able to quantitatively image endogenous survivin mRNA and release the loaded drug in a manner that depends on the survivin expression level in tumor cells. The authors further show that this novel drug delivery system may be used to minimize potential cytotoxicity encountered by many existing small-molecule drugs in cancer therapy.

Full text of DOI:10.1002/smll.201700569

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Imaging
Simultaneous Imaging of Endogenous Survivin mRNA
and On-Demand Drug Release in Live Cells by Using
a Mesoporous Silica Nanoquencher
Peiyan Yuan, Xin Mao, Kok Chan Chong, Jiaqi Fu, Sijun Pan, Shuizhu Wu,
Changmin Yu,* and Shao Q. Yao*
The design of multifunctional drug delivery systems capable of simultaneous
target detection, imaging, and therapeutics in live mammalian cells is critical for
biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs)
chemically modified with a small-molecule dark quencher, followed by sequential
drug encapsulation, MSN capping with a dye-labeled antisense oligonucleotide, and
bioorthogonal surface modification with cell-penetrating poly(disulfide)s, the authors
have successfully developed the first mesoporous silica nanoquencher (qMSN),
characterized by high drug-loading and endocytosis-independent cell uptake, which
is able to quantitatively image endogenous survivin mRNA and release the loaded
drug in a manner that depends on the survivin expression level in tumor cells. The
authors further show that this novel drug delivery system may be used to minimize
potential cytotoxicity encountered by many existing small-molecule drugs in cancer
therapy.
cargos.[2] In addition to key properties such as high cargo
1. Introduction
loading capacity, good biocompatibility, and easy surface
In recent years, nanotechnology has attracted much atten- functionalization, the inert architectures of these nanomate-
tion in biomedical research. Among various nanomaterials, rials further endow the encapsulated cargo with good intra-
mesoporous silica nanoparticles (MSNs) are widely used cellular stability. By choices of different gatekeepers to cap
in drug delivery and diagnostics/bioimaging.[1] These orga- the surface of cargo-loaded nanoparticles, MSNs may facili-
nosilicon materials could be readily prepared in the typical tate delivery and controlled release of drugs exclusively to
range of 50–200 nm to possess periodic pores of several the intended cells and tissues, without provoking adverse side
nanometers, capable of efficiently encapsulating a variety of effects commonly associated with small-molecule drugs.[3] To
date, numerous types of “gated” MSNs have been reported.[4]
Among them, nucleic acid capped MSNs are highly attractive
Dr. P. Yuan, X. Mao, K. C. Chong, Dr. J. Fu, S. Pan,
as they offer a sequence-encoded universal strategy of drug
Prof. S. Q. Yao
release that may be triggered by any endogenous DNAs/
RNAs expressed in tumor cells or tissues.[5] By using chemi-
Department of Chemistry
National University of Singapore
cally modified antisense oligonucleotide (ASO)-capped
MSNs, we recently reported a drug delivery system capable
of intracellular controlled release of drugs in a manner
dependent upon the endogenous expression of microRNAs
(i.e., well-known small noncoding regulatory RNAs).[6]
Whether or not a similarly gated system can effect such
on-demand drug release by other endogenous genetic mate-
rials (i.e., mRNAs) is, however, unknown. Furthermore, the
3 Science Drive 3, 117543 Singapore, Singapore
E-mail: chmyaosq@nus.edu.sg
Prof. S. Wu, Dr. C. Yu
College of Materials Science and Engineering
South China University of Technology
510640 Guangzhou, China
E-mail: yucm@scut.edu.cn
DOI: 10.1002/smll.201700569
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cellular uptake of these nanoparticles was facilitated by cova- nanoquencher (qMSN), obtained by conjugating a small-
lent modification of the MSN surface with cell-penetrating molecule fluorescence dark quencher (Black Hole Quencher
peptides, which caused severe endolysosomal trapping and (BHQ), BHQ-2) to amine-functionalized MSNs (MSN-NH2).
led to slow/poor cytosolic release of MSNs.[7] Such problems BHQ-2 was chosen because its absorption spectrum
are even more pronounced with larger-size MSNs (>100 nm) (550–650 nm) overlaps well with the emission spectrum of
which are needed to ensure high drug-loading capacity.[1,2] It tetramethylrhodamine (TMR). Similar BHQ-2-modified
was found that prolonged incubations with cells were needed nanoquenchers were previously reported as fluorescent sen-
in order to achieve sufficient intracellular uncapping of such sors to detect endogenous enzymatic activities,[16] but never
MSNs (>6 h) and their subsequent drug release (12–24 h).[6,8] as dual-imaging drug delivery systems. By optimizing the
Such a time scale is clearly unacceptable if the MSNs are amount of BHQ-2 modified on the surface of positively
intended for theranostic applications to image the target, charged nanoparticles, followed by drug encapsulation
drug release, and/or the drugs biological effects.[8]
and capping with a negatively charged TMR-labeled ASO
Nucleic-acid-based reagents capable of mRNA imaging (via electrostatic interaction), we successfully synthesized
and quantification are widely available, but a few are able to ASO-Drug-qMSN that possessed both good fluorescence
enter mammalian cells on their own, and thus offer limited quenching efficiency (QE) (≈94%) and high drug-loading
utilities for intracellular applications.[9] Recent advances in capacity (>58 mol g−1 for Doxorubicin, or Dox). Typical
nanotechnology have fueled the development of gold nano- AuNPs and MSNs would possess one of these two proper-
particle (AuNP)-coupled fluorescent DNA probes, which ties, respectively; but not both.[1,2,10] Deviated from our pre-
are suitable for real-time and quantitative imaging of endog- vious work, in which chemically modified ASO was used
enous mRNAs.[10] The many favorable properties that these both as a gatekeeper and a microRNA inhibitor by antisense
AuNPs possess, including cell permeability (albeit mostly technology,[6] the survivin ASO used in the current work
with endocytosis uptake mechanism),[10a] strong nucleic-acid- (Scheme 1a), with the exception of TMR labeling, was not
binding, resistance to degradation, and distance-dependent chemically modified, and therefore was capable of binding
fluorescence quenching, make them well-suited for the design to survivin mRNA via sequence complementarity, but not
of various fluorescence Turn-ON nanosensors capable of affecting its endogenous expression level.[17] Survivin is an
responding to different endogenous biomolecules and/or important tumor biomarker that promotes cell prolifera-
stimuli.[11] One major shortcoming of existing AuNP-based tion, and high levels of survivin (at both mRNA and protein
mRNA nanosensors lies in their limited intracellular detec- levels) are found in most cancer cells.[18]
tion sensitivity, which stems from the intrinsically low probe-
Another important feature of above qMSN was that,
loading capacity due to small nanoparticle sizes (≤10 nm), as after ASO capping, an additional coating of cell-penetrating
well as slow/poor cytosolic release due to endocytosis.[10,12] poly(disulfide)s (CPD) was introduced to the outermost layer
Introduction of enzyme-free signal amplification techniques, of the nanoparticles (giving CPD-ASO-Drug-qMSN). This was
such as hybridization chain reaction into these nanosen- done by treatment of the qMSN surface with N3-APS (ammo-
sors, has presumably led to the improved detection limit.[13a] nium persulfate), followed by successive bio-orthogonal liga-
Another major problem with AuNP-based mRNA biosensors tion with TCO-PEG12-DBCO and Tz-CPD (Scheme 1b).[19]
is their limited ability to be doubled as effective drug delivery We found this “layer-by-layer” surface modification was neces-
systems, unlike MSNs.[10b] Consequently, theranostic nano- sary to ensure the resulting nanoparticles entered the cytosol
particles capable of both quantitative imaging of endogenous of mammalian cells via endocytosis-independent pathways
mRNAs and efficient drug delivery (and on-demand release) (Step I) followed by glutathione (GSH)-assisted rapid depo-
are, to our knowledge, unavailable at present.[14]
lymerization of CPD (<5 min;[20] Step II), before being exposed
Herein, we report the successful development of new to any endogenous survivin mRNA. Compared to other endo-
theranostic MSNs that achieved not only intracellular cytic nanoparticles,[10] CPD-conjugated nanoparticles were
controlled release of an encapsulated drug in a manner previously shown to be rapidly and efficiently uptaken by
dependent upon the endogenous expression level of a target mammalian cells without endolysosomal trapping,[21] and we
mRNA, but also quantitative imaging of both the target (i.e., were hopeful that this important quality would further boost
mRNA) and the drug release process (Scheme 1). It may also the mRNA imaging capability of these qMSNs. Next, in the
be used to minimize potential cytotoxicity encountered by presence of cytosolic survivin mRNA, the qMSN-bound, fluo-
many existing small molecule drugs in cancer therapy.[3] The rescently quenched TMR-ASO would undergo hybridization
ability to image endogenous mRNAs in live mammalian cells and get detached from the positively charged qMSN surface,
and tissues is important in revealing the expression, localiza- resulting in an immediate and proportional release of fluores-
tion, and dynamics of these essential cellular molecules.[15]
cence signals (Step III). Concurrently, the dissociation of TMR-
ASO gatekeeper would also cause the uncapped qMSN to
release the loaded drug intracellularly (Step IV).
Detailed synthesis of CPD-ASO-Drug-qMSN and control
MSNs are provided in Figure S1 and S2 in the Supporting
2. Results and Discussion
Overall strategy of our newly developed theranostic MSNs Information. The chemical, biochemical, and physical prop-
is shown in Scheme 1. A key feature of these multifunction erties of these nanoparticles were characterized by transmis-
MSNs for simultaneous imaging of endogenous mRNAs and sion electron microscopy (TEM), Brunauer–Emmett–Teller
on-demand drug release was the use of a mesoporous silica (BET), Fourier transform infrared (FTIR), Zeta potential,
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Scheme 1. a) Overall strategy for the preparation of CPD-ASO-Drug-qMSN and subsequent cellular uptake via endocytosis-independent pathways
(I), GSH-assisted CPD depolymerization (II), imaging of endogenous survivin mRNA (III), and on-demand release of drug (IV). b) Scheme showing
the chemistries used for layer-by-layer surface qMSN modification with N3, SPAAC ligation between N3 and DBCO of TCO-PEG12-DBCO, and finally
TCO-tetrazine ligation between TCO and the tetrazine of Tz-CPD.
and other measurements as previously described the presence of 2 × 10−6 m of synthetic survivin mRNA and a
(Figure 1 and Figure S3, Supporting Information).[6] Briefly, calculated detection limit of 1.3 × 10−9 m, with a linear corre-
highly monodispersed MSN-NH2 was first prepared (mean lation at λem = 580 nm in the range of 0 to 100 × 10−9 m (inset
size: 120.7 6.4 nm) and possessed a uniform pore size of in Figure 2b). As shown in Figure 3a, while most of TMR flu-
2.6 nm. Subsequently, as-synthesized BHQ-2-APS was intro- orescence from detached TMR-ASO of the nanosensor upon
duced as previously reported.[16] We optimized the amount addition of synthetic survivin mRNA could be recovered in
of BHQ-2 modified on MSNs to obtain high quenching effi- <2 h, no obvious fluorescence increase was observed with a
ciency for TMR-ASO after capping, and a 94% quenching non-complimentary mRNA (control 18A RNA), even after
efficiency was achieved with an estimated 10.67 µmol g−1 prolonged incubation (24 h; Figure 3a). Similar fluorescence
of BHQ-2 loading, as shown in Figure S4 in the Supporting Turn-ON profiles were obtained with ASO-qMSN incubated
Information. The amount of capping TMR-ASO was deter- with different amounts of total RNA isolates of HeLa cells,
mined by fluorescence measurement to be 18.46 µmol g−1, which are known to have elevated endogenous survivin
and the corresponding controlled release experiments were mRNA expression (Figure S5a, Supporting Information).[13]
similarly done in the presence of synthetic survivin mRNA These results indicate our newly developed nanosensor has
under in vitro conditions (Figure 2a); while an ASO-qMSN the potential to sensitively and quantitatively detect and
solution displayed very weak fluorescence, its fluorescence image endogenous survivin mRNA in a sequence-specific
intensity increased proportionally upon addition of syn- manner with relatively short response time.
thetic survivin mRNA, indicating successful detachment of
Next, the intrinsically fluorescent Dox (an anticancer
TMR-ASO from the qMSN surface as a result of sequence- drug) was loaded into ASO-qMSN to monitor the con-
matched recognition by the synthetic survivin mRNA. We trolled release process of the drug in response to survivin
observed a 5.2-fold Turn-ON fluorescence of ASO-qMSN in mRNA. The amount of loaded Dox as well as its subsequent
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for 24 h (Figure S3e,f, Supporting Information); fluorescence
increases from either TMR or Dox channel (indicator of
possible TMR-ASO degradation or premature Dox release,
respectively) was not observed, thus confirming that, in our
subsequent live-cell experiments, successful fluorescence
Turn-ON and ensuing drug release were caused by endog-
enous target recognition and not serendipitous nuclease
degradation of the TMR-ASO gatekeeper. Previously, it was
observed that the high-affinity electrostatic assembly of DNA
on the cationic surface of MSNs probably cause high steric
hindrance for nuclease to interact with DNA, thus effectively
protecting DNA against potential degradation.[6,13]
We next determined the endogenous mRNA imaging
property of these nanoparticles in live mammalian cells. An
additional coating of CPD was introduced to TMR-ASO-
capped qMSN, as earlier described, giving CPD-ASO-qMSN
(no drug) and CPD-ASO-Drug-qMSN (with a drug). The
resulting nanoparticles showed negligible aggregation in
Dulbecco’s Modified Eagle Medium (DMEM) after 30 h
incubation (Figure 1d). Cellular uptake and survivin mRNA
imaging capability of CPD-ASO-qMSN in HeLa cells were
next examined by using confocal laser scanning microscopy
(CLSM). In order to confirm the endocytosis-independent
cell uptake of CPD modified MSNs, detailed uptake studies
Figure 1. TEM image of a) MSN-NH2 without CTAB and b) ASO-qMSN
without CTAB. c) Zeta potentials of different MSNs. d) DLS size
measurements of CPD-ASO-qMSN incubated in DMEM medium for
different periods of time (0, 3, 18, 30 h).
release from the resulting ASO-Dox-qMSN was quantita- were conducted by using FITC-labeled MSNs as model
tively determined by fluorescence measurements of Dox cargos (Figure S6, Supporting Information). Unlike control
(Figure 3 and Figures S4 and S5, Supporting Informa- MSNs without CPD modification (FITC-MSN-NH2), cell
tion), and the Dox loading capacity was determined to be uptake of CPD-FITC-MSN was not sensitive to treatment by
58.85 µmol g−1 (Figure S4d, Supporting Information). Time- most endocytosis inhibitors. Treatment of cells with a thiol-
dependent Dox release from ASO-Dox-qMSN was observed mediated translocation inhibitor, 5,5′-dithiobis-2-nitroben-
in the presence of 0.3–2.0 × 10−6 m of synthetic survivin zoic acid, on the other hand, caused significant inhibition of
mRNA, which showed >80% Dox was successfully released CPD-FITC-MSN uptake. Moreover, no significant endolyso-
after 24 h incubation (at 37 °C), while <10% of drug leakage somal trapping was observed when CPD-FITC-MSN entered
was detected during the same period with either a “wrong” cells. Such results are consistent with our previous studies,[21]
mRNA (e.g., 2.0 × 10−6 m of 18A RNA) or no mRNA. Longer and again confirm the endocytosis-independent, thiol-medi-
incubation and increasing amounts of synthetic survivin ated cell uptake mechanism of CPD-modified MSNs.[20]
mRNA both led to proportional increases of Dox release After that, RGD-ASO-qMSN, which were prepared by cap-
(Figure 3b), indicating this “smart” drug delivery system ping qMSN with TMR-ASO followed by coating with an
would enable dose-dependent, controlled release of a small arginylglycylaspartic acid (RGD) peptide and were expected
molecule drug in tumor cells in response
to important endogenous tumor bio-
markers such as survivin mRNA. A linear
relationship between the amount of Dox
release (x-axis) and the fluorescence inten-
sity (y-axis) from the detached TMR upon
addition of different doses of synthetic
survivin mRNA and different incubation
time further showed (Figure 3c,d), while
our qMSN was designed to image endog-
enous survivin mRNA, the TMR fluores-
cence from such experiments could also be
Figure 2. a) Fluorescence spectra (λex = 545 nm) of ASO-qMSN (0.1 mg mL−1 in PBS) incubated
with varying concentrations of synthetic survivin mRNA (0 × 10−9 m, 12.5 × 10−9 m, 25 × 10−9 m,
50 × 10−9 m, 100 × 10−9 m, 200 × 10−9 m, 500 × 10−9 m, 1000 × 10−9 m, 2000 × 10−9 m)
at 37 °C for 2 h. Inset: Photos of the ASO-qMSN sample (boxed in Figure S4b, Supporting
Information) taken under UV (λex = 365 nm), after incubation with (left) or without (right)
synthetic survivin mRNA. b) Plot of fluorescence intensity of ASO-qMSN (0.1 mg mL−1 in PBS;
37 °C for 2 h) versus target concentrations (synthetic survivin mRNA: black; control 18A RNA:
red). Inset: plot of fluorescence intensity (λem = 580 nm) of ASO-qMSN versus synthetic
used to quantitatively monitor the relative
amount of the encapsulated drug released
from the nanoparticles, including those
without intrinsic fluorescence. Finally, the
biochemical stability of ASO-Dox-qMSN
in cellular environments was tested in a
model experiment by treating the nano-
particles with DNAase I (up to 5 U mL−1) survivin mRNA (0 to 100 × 10−9 m).
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CPD-18C-qMSN treated cells (middle and
bottom panels), respectively. All results
were further quantified (Figure S7, Sup-
porting Information) Next, we ascertained
whether these nanosensors could be
used to quantitatively image and differ-
entiate survivin mRNA levels expressed
in various mammalian cells. In addition
to HeLa cells, which are known to have
a high survivin expression,[13,18] MCF-7
cells (medium survivin expression) and
MCF-10A cells (low/minimum survivin
expression) were treated with CPD-ASO-
qMSN and imaged (Figure 4c); strong,
medium, and weak/no TMR fluorescence
signals were detected in cytosols of the
treated cells, and these imaging-based
results correlated well with the relative
expression levels of endogenous survivin
mRNA concurrently determined by real-
time quantitative PCR (qPCR; Figure S9,
Supporting Information). These survivin
mRNA-responsive TMR fluorescence
changes were further quantified using the
flow cytometry (Figure 4c, right). We fur-
ther expanded this quantitative imaging-
based survivin mRNA detection method
to other mammalian cell lines (i.e., A549,
A431, PC3, HepG2, HEK 293T). Instead
of quantifying survivin mRNA at single-
cell level, which might not be as accurate
due to cell-to-cell variations, we used an
Figure 3. a) Time-dependent fluorescence increase of ASO-qMSN (0.1 mg mL−1 in PBS at
37 °C, λem = 580 nm) upon addition of different targets (target concentrations: 2.5 × 10−6 m,
synthetic survivin mRNA: black; control 18A RNA: red). b) Time-dependent Dox release from
ASO-Dox-qMSN (0.1 mg mL−1 in PBS at 37 °C) upon incubation with synthetic survivin mRNA
(0, 0.3, 1.0, 2.5 × 10−6 m). c) Plot of fluorescence intensity (λem = 580 nm) of ASO-qMSN
versus % Dox release (λex = 488 nm, λem = 558 nm) after 2 h incubation (at 37 °C) with
different amounts of synthetic survivin mRNA. d) Time-dependent plot of fluorescence
intensity of released TMR-ASO versus released Dox after ASO-Dox-qMSN (0.1 mg mL−1 in PBS)
was incubated with 2.5 × 10−6 m of synthetic survivin mRNA.
to be uptaken by mammalian cells via endocytosis,[6] were imaging-based high-content screening (HCS) system to ana-
used as another type of control nanoparticles to confirm lyze a large pool of nanosensor-treated cells (Figure 4d and
our earlier hypothesis, that is, the rapid, efficient, and endo- Figure S10 in the Supporting Information); a nearly linear
cytosis-independent cell uptake properties expected from relationship between qPCR results and average TMR sig-
CPD-ASO-qMSN would render them more superior for nals obtained from the HCS experiment further confirmed
live-cell quantitative imaging of endogenous survivin mRNA our newly developed nanosensors could indeed be used to
than traditional endocytic nanosensors.[10a] CPD-18C-qMSN, quantitatively image endogenous survivin mRNA at single-
prepared by capping qMSN with TMR-18C (a naive gate- cell level or with an ensemble of cells at different spatial
keeper; Scheme 1a) followed by CPD coating, were designed resolutions.
to validate that, only a properly gated nanosensor (i.e., CPD-
The on-demand drug release property of these qMSNs in
ASO-qMSN) would enable successful imaging of endog- response to endogenous survivin mRNA was next assessed.
enous survivin mRNA. CLSM of live HeLa cells treated with Previously reported survivin mRNA nanosensors do not
CPD-ASO-qMSN showed visible TMR fluorescence in as have such drug-delivery capability.[10,13] Two recently devel-
little as 20 min of individual cells (Figure 4a), indicating suc- oped MSNs only responded to microRNA or endogenous
cessful cell uptake of the nanosensor and subsequent target GSH and did not have any mRNA imaging capability.[6,21]
recognition/TMR-ASO release. The TMR fluorescence sig- As shown in Figure 4e,f, CPD-ASO-Dox-qMSN was used
nals continued to increase over the course of 4 h, as more to treat HeLa cells, followed by time-dependent live-cell
nanosensors reached the cytosol where endogenous sur- imaging under both TMR (red) and Dox channels (green); by
vivin mRNA resided, resulting in the release of free TMR- simultaneously imaging both the endogenous survivin mRNA
ASO, which led to a further increase in TMR fluorescence. (TMR channel) and on-demand Dox release (Dox channel),
Side-by-side comparison of HeLa cells treated with control we were able to closely monitor the sequential uncapping of
nanoparticles showed even more pronounced differences TMR-ASO and release of encapsulated Dox from the qMSN.
in their single-cell mRNA imaging capability (Figure 4b, Similar to earlier observations (Figure 4a and Figure S8, Sup-
4 h incubation); very strong TMR fluorescence was detected porting Information), TMR fluorescence started to develop
throughout the cytosol of CPD-ASO-qMSN-treated cells in 20 min, at which point no obvious Dox fluorescence was
(top panels), while significantly weaker and no TMR fluo- detected. At 40 min, the Dox fluorescence appeared, indi-
rescence signals were detected in RGD-ASO-qMSN and cating a successful drug release. Further incubation led to
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Figure 4. a) Real-time 3D projections of z-stack images at 90° view angles (step size = 0.163 µm) of live HeLa cells incubated with 10 µg mL−1
CPD-ASO-Dox-qMSN for different lengths of time before image acquisition (0, 20, 40, 60, and 80 min). (Dox): green; (TMR): red; (Hoechst): blue.
b) 3D projections of z-stack images at different view angles (0°, 45°, 90°, −45°) of HeLa cells treated with CPD-ASO-qMSN, RGD-ASO-qMSN and
CPD-18C-qMSN (10 µg mL−1; 4 h). Cells were stained with Hoechst (in blue). Turn-ON fluorescence from the TMR channel was colored in red. c) CLSM
images of different mammalian cells treated (left) with and (middle) without CPD-ASO-qMSN (10 µg mL−1; 4 h). (right) Flow cytometric results of
different mammalian cells treated with CPD-ASO-qMSN (red, 10 µg mL−1, for 4 h), cells without any qMSN treatments were used as control (black).
(x-axis): relative TMR fluorescence; (y-axis): cell count. d) Fluorescence quantification in different cell lines by HCS versus their endogenous survivin
mRNA expression levels determined by qPCR. Cells were incubated with CPD-ASO-qMSN (10 µg mL−1; 4 h). Error bars were obtained by analysis
of triplicate tests. e) CLSM images of HeLa cells treated with 10 µg mL−1 CPD-ASO-Dox-qMSN after different incubation time (2, 4, 12, 24 h). f) 3D
projections of z-stack images at 90° view (step size, 0.163 µm) of HeLa cells treated with CPD-ASO-Dox-qMSN (10 µg mL−1; 24 h). Scale bar = 20 µm.
concomitant increases in both TMR and Dox fluorescence as confirmed by flow cytometry (Figure S12, Supporting Infor-
more TMR-ASO uncapping and drug release occurred. This mation). We noted this intracellular drug-release profile was
process continued until at ≈24 h, when both TMR and Dox similar to that of earlier in vitro results (Figure 3), and that
signals reached saturation. 3D projections of z-stack images TMR fluorescence was detected prior to Dox fluorescence
of these treated cells clearly showed the subsequent nuclear clearly indicate the role of TMR-ASO as a gatekeeper in pre-
localization of released Dox, while the TMR fluorescence sig- venting premature leakage of Dox.As expected, neither TMR
nals remained mostly in the cytosol (Figure 4f and Figure S11, nor Dox fluorescence was detected in control cells treated
Supporting Information). We observed apparent rounding with CPD-18C-Dox-qMSN over 24 h (Figure S13, Supporting
in the cells indicative of apoptosis, which was subsequently Information). To further confirm that our delivery system was
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Figure 5. a) CLSM images of HeLa cells treated with YM155 (0 × 10−9 m, 2.5 × 10−9 m, 5 × 10−9 m; 48 h) followed by mRNA imaging with CPD-ASO-
qMSN (10 µg mL−1; 4 h). b) Flow cytometric results of HeLa cells treated with different amounts of YM155 (0 × 10−9 m: red; 2.5 × 10−9 m: blue;
5 × 10−9 m: green) followed by incubation with CPD-ASO-qMSN (10 µg mL−1, for 4 h). HeLa cells without any qMSN treatments were used as control
(black). (x-axis): relative TMR fluorescence; (y-axis): cell count. c) qPCR determination of endogenous survivin mRNA expression from cells in (a).
d) XTT cell viability test (black bars) and endogenous surviving mRNA expression levels (red bars) of HeLa (top graph) or MCF-10A (bottom graph)
cells treated with different qMSNs (50 µg mL−1; 24 h). Control: DMSO-treated cells. Note: data in y-axis of each graph were normalized against
“control” which was set as either 100% (cell viability) or 1 (survivin mRNA expression). e) WB analysis of cells in (d), with anti-cleaved caspase-3
and anti-tubulin (loading control), respectively. Scale bar = 20 µm. Error bars were obtained by analysis of triplicate tests.
able to achieve survivin mRNA-responsive controlled drug target imaging and dose-dependent release of YM155 intra-
release as well as quantitatively monitor the released amount cellularly with an auto-feedback inhibition mechanism, that
of the encapsulated drug, cell lines with different survivin is, the more endogenous survivin mRNA are detected in the
mRNA expression level were imaged after the treatment nanoparticle-treated tumor cells, the more YM155 would be
with CPD-ASO-Dox-qMSN (Figure S8c and S14, Supporting released to suppress its expression. As shown in Figure 5a,b,
Information). Both TMR and Dox fluorescent signals cor- HeLa cells pretreated with different dosages of YM155 (0,
respondingly changed with the endogenous survivin mRNA 2.5, 5 × 10−9 m for 48 h) followed by endogenous survivin
level, indicating this new developed theranostic system can mRNA imaging with CPD-ASO-qMSN clearly showed dose-
be utilized to simultaneously endogenous image both the dependent down-regulation of survivin mRNA with corre-
target and drug release process.
sponding decreases in TMR fluorescence signals of treated
Most small molecule drugs have side effects caused by off- cells, and these results were in good agreement with the
targets and other issues. While advanced chemical tools have qPCR data (Figure 5c and Figure S14, Supporting Informa-
been developed that enable proteome-wide identification of tion). Since most drugs including YM155 are not fluorescent
potential cellular targets of bioactive compounds,[3,22–29] they and cannot be imaged directly, our earlier results showing a
do not make a drug less cytotoxic, unless new analogs are linear relationship between Turn-ON TMR signals and the
identified/tested. In order to side-step such off-target issues, relative amount of a released drug (Figure 3) mean that
we explored alternative “smart” drug delivery methods above-detected TMR signals also served as a reliable indi-
capable of delivering cytotoxic drugs to specific cells/tissues cator to assess the relative amount of intracellularly released
and releasing them at the right dosage. YM155 is an imidazo- YM155 in the following experiments. First,YM155 was loaded
lium-based compound that shows potent antitumor activities into CPD-ASO-qMSN to give CPD-ASO-YM155-qMSN.
by effectively suppressing the endogenous survivin mRNA Next, these drug-loaded nanoparticles were incubated with
expression from a variety of cancer cells.[30,31] We wondered HeLa cells followed by XTT viability analysis (to measure
if our newly developed theranostic nanoparticles could be cell killing effect), qPCR (to measure endogenous survivin
used to deliver YM155 into cancer cells, thus enabling both mRNA expression), flow cytometry analysis and western
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blotting (WB) analysis (to measure cell apoptosis). Control Antibodies were purchased from following vendors: rabbit anti-
experiments with MCF-10A cells which have minimum sur- cleaved caspase-3 (#9664, Cell signaling Technology), rabbit anti-
vivin mRNA expression were concurrently carried out to β-tubulin (#ab6064, abcam), and goat anti-rabbit IgG (#A11008,
assess whether CPD-ASO-YM155-qMSN was indeed able Invitrogen). Western blotting was performed by using the ECL Plus
to minimize the cytotoxicity of YM155 by effectively killing Western Kit (GE Healthcare), and imaged on an ImageQuant LAS
ONLY survivin-overexpressing cancer cells, such as HeLa 500 system (GE). All regents were of analytical grades and used
as received without further purification, unless otherwise indi-
cated. All aqueous solutions were prepared by using diethyl pyro-
carbonate (DEPC)-treated ultrapure water from a Milli-Q system.
Phosphate buffered saline (10 × 10−3 m, pH 7.4) was diluted from
10× PBS buffer, containing 136.7 × 10−3 m NaCl, 2.7 × 10−3 m KCl,
8.7 × 10−3 m Na2HPO4, and 1.4 × 10−3 m KH2PO4. All oligonucleo-
tides were purchased from Integrated DNA Technologies (Singa-
pore). Their sequences are as follows:
cells. As shown in Figure 5d,e and Figure S15, Supporting
Information, while both cancer cells were effectively killed
by free YM155 (indicative of the drug’s intrinsic cytotoxicity),
Only HeLa cells were killed by CPD-ASO-YM155-qMSN
(top graph in Figure 5d; black bars); we observed >50% sup-
pression of HeLa cell growth in the presence of these drug-
loaded nanoparticles, while similarly treated MCF-10A cells
showed negligible growth inhibition (bottom graph; black
bars). The cell growth inhibition profiles corroborated well
with corresponding changes in the survivin mRNA expres-
sion levels (red bars in Figure 5d), therefore unequivo-
cally confirming our delivery system and its controlled drug
release were indeed survivin mRNA-responsive. Finally, WB
analysis further confirmed that, only YM155-loaded nano-
particles capped with TMR-ASO (e.g., CPD-ASO-YM155-
qMSN), but not any other control nanoparticles, were able
to induce caspase-3-activated apoptosis in HeLa cells (top
gels in Figure 5e, and Figure S16, Supporting Information);
no apoptosis was detected in similarly treated MCF-10A cells
(bottom gels).
Probe
RNA or DNA sequence (5′−3′)
Synthetic survivin mRNA
target
rArArU rUrCrA rCrArG rArArU rArGrC
rArCrA
Antisense oligo (TMR-ASO)
against survivin mRNA
TMR-TGT GCT ATT CTG TGA ATT
18C naïve oligo (TMR-18C)
as negative gatekeeper
TMR-CCC CCC CCC CCC CCC CCC
Non-complementary control rArArA rArArA rArArA rArArA rArArA
synthetic target (18A) rArArA
*r(A/U/C/G) and (A/T/C/G) denote the corresponding ribonucleotides and deox-
yribonucleotides, respectively.
Fluorescence spectra were collected on a Horiba FluoroMax-4
fluorometer. Ultraviolet–visible (UV–vis) extinction spectra were
measured on a Shimadzu UV-2450 spectrophotometer. TEM
images were obtained on a JEOL 2010 transmission electron
microscope at an accelerating voltage of 200 kV. Zeta-potential,
particle size and distribution were performed on a Malvern Nano-
ZS90. Flow cytometric analysis was carried out on a BD Accuri C6
cell analyzer. The confocal cell images were obtained on a Leica
TCS SP5 X Confocal microscope equipped with a 63× water immer-
sion objective. 3D projection of z-stack images were collected on
a FV1000-X81 confocal microscope (Olympus) equipped with a
water immersion objective (60×). HCS were acquired with an In-
Cell Analyzer 2200 (GE Healthcare) equipped with a solid-state,
multi-wavelength illuminator. Images were processed and quanti-
fied with In-Cell Developer Toolbox software 1.9.2. Real-time qPCR
was carried out on a Bio-Rad MyiQ RT-PCR thermo cycler.
Synthesis of Tz-CPD: The Tz-CPD polymer used in this study was
synthesized and characterized according to previously published
procedures (Figure S1a, Supporting Information).[20a] The mole-
cular weight of the Tz-CPD polymer was determined to be ≈20 KDa
by analytical gel permeation chromatography (GPC), with an esti-
mated stock concentration of ≈200 × 10−6 m. The N3-RGD pep-
tide was synthesized by standard solid-phase peptide synthesis
according to previously reported procedures.[6]
3. Conclusions
In conclusion, by using mesoporous silica nanoparticles chem-
ically modified with a dark quencher, followed by sequential
drug encapsulation, MSN capping with a dye-labeled anti-
sense oligonucleotide and bioorthogonal surface modification
with cell-penetrating poly(disulfide)s, we have successfully
developed the first mesoporous silica nanoquencher charac-
terized by high drug loading and endocytosis-independent
cell uptake, and was able to quantitatively image endogenous
survivin mRNA and release the loaded drug in a manner that
depended on the survivin expression level in tumor cells. This
novel theranostic system unifies endogenous imaging of both
the target and drug release process, and on-demand drug
release into a single platform. It may also be used to minimize
potential cytotoxicity encountered by many existing small
molecule drugs in cancer therapy. Finally, the use of qMSN
for quantitative imaging of endogenous mRNA by taking
advantage of sequence-based hybridization/de-hybridization
of gated, fluorescently labeled ASO for binding/recognition
of endogenous mRNA is conceptually novel, and should be
universally applicable for live-cell imaging of many other bio-
logically relevant mRNAs.
Synthesis of BHQ-2-APS, N3-APS and Tetrazine-APS: As shown
in Figures S1b and S1c in the Supporting Information, APTES
(8.6 mg) was reacted with 50 µL of BHQ-2 NHS ester stock solu-
tion in anhydrous DMF (0.01 mg mL−1) containing 2% diisopro-
pylethyalamine (DIPEA) overnight, as previously described.[16]
Subsequently, this freshly prepared BHQ-2-APS stock was used in
the next-step MSN modification without any further purifications.
4. Experimental Section
Materials: Tetraethylorthosilicate (TEOS), N-cetyltrimethylam-
monium bromide (CTAB), 3-aminopropyltriethoxysilane (APTES),
and sodium hydroxide were purchased from Sigma. YM155 was
obtained from Selleckchem. Black hole quencher-2 (BHQ-2)
NHS ester was purchased from Biosearch Technologies (USA).
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For the reaction stock of N3-APS, first, 6-azidohexanoic acid succin- and the reaction was stirred overnight, followed by centrifugation
imidyl ester was synthesized according to published procedures and washing with water (3 times). To load the drug (i.e., doxoru-
as the followings.[4] 6-Bromohexanoic acid (1.50 g, 7.67 mmol) bicin, or Dox), 100 µL of the obtained TCO-qMSN (redispersed
was first dissolved by stirring in anhydrous DMF (5 mL), followed in H2O, 1.0 mg mL−1) was incubated with 20 µL of 50 × 10−3
m
by addition of sodium azide (1.0 g, 15.4 mmol) into the solu- Dox stock solution (dissolved in H2O) for 24 h, followed by cen-
tion, and the mixture was stirred at 85 °C for 3 h. The mixture was trifugation, washed with water (2 times) and resuspension in PBS
diluted in dichloromethane (50 mL) and washed with hydrochloric (50 µL). The capping of the nanoparticles with ASO (or a control
acid (50 mL, 0.1 m). The organic layer was extracted, washed with oligo) was next done by addition of 6.0 µL of the oligo stock solu-
hydrochloric acid (0.1 m), dried over anhydrous sodium sulfate, tion (100 × 10−6 m), followed by incubation at 37 °C for 2 h. Upon
and concentrated in vacuo to yield crude 6-azidohexanoic acid centrifugation and washing with water (3 times), the resulting ASO-
(1.0 g, 83% yield) as a yellow oil-like solution. 1-Ethyl-3-(3-dimeth- Dox-qMSN were redispersed in PBS (100 µL). Finally, 5.0 µL of the
ylaminopropyl) carbodiimide (720 mg, 3.76 mmol) was added to earlier-prepared Tz-CPD stock (50 m) was added to the nanoparticle
this crude 6-azidohexanoic acid (590 mg, 3.76 mmol) dissolved solution (1.0 mg mL−1, 50 µL), and the mixture were incubated at
in CHCl3/DMF (9:1, 1 mL), followed by addition of N-hydroxysuc- room temperature for 1 h. Upon centrifugation and washing with
cinimide (432 mg, 3.76 mmol). The reaction mixture was stirred at water (2 times), the resulting nanoparticles were redispersed in
room temperature for 18 h, then diluted with CH2Cl2 (50 mL) and PBS (50 µL), giving CPD-ASO-Dox-qMSN (1.0 mg mL−1). CPD-ASO-
washed with dilute hydrochloric acid (50 mL, 0.1 m). The organic qMSN loaded with other drugs (i.e., YM155; 50 × 10−3 m stock in
layer was extracted, rinsed with hydrochloric acid, and washed DMSO), or no drug, were similarly prepared. Nanoparticles capped
with aqueous sodium bicarbonate and brine. The organic layer was with a control oligo (i.e., TMR-18C) were similarly prepared, except
dried over anhydrous sodium sulfate and concentrated in vacuo to TMR-18C was used in place of survivin TMR-ASO during the cap-
yield a yellowish solution which was further purified by flash chro- ping step.
matography (3:7 ethyl acetate/dichloromethane) to give the pure
Preparation of RGD-ASO-qMSN: The RGD-ASO-qMSN, used
6-Azidohexanoic acid succinimidyl ester (600 mg, 63% yield). as control nanoparticles to evaluate the effect of CPD in facili-
Characterizations of this product were in good agreement with pre- tated cell uptake and improved imaging capability of qMSN,
viously reported data.[32] 1H NMR (300 MHz, CDCl3): δ 1.54–1.38 were prepared as the following (Figure S2b, Supporting Informa-
(m, 2H), 1.67–1.62 (m, 2H), 1.82–1.77 (q, 2H), 2.57–2.47 (q, 2H), tion). 1.0 mg of above-synthesized qMSN without CTAB was first
2.74 (br s, 4H), 3.24–3.19 (t, J = 6.5 Hz, 2H). Subsequently, the suspended in 5 mL dry toluene. Tetrazine-APS (4 mg) was sub-
reaction stock of N3-APS was prepared by mixing 0.6 µmol of sequently added and the mixture was stirred at 50 °C under a
6-Azidohexanoic acid succinimidyl ester with APTES (8.6 mg) in nitrogen atmosphere for 6 h. The nanoparticles were centrifuged
anhydrous DMF (50 µL) in the presence of 2% DIPEA overnight, and washed with methanol and DI water, then freeze-dried over-
and used in the next-step MSN modification without any further night to obtain Tz-qMSN. Upon resuspension in 0.2 mL DMF (final
purifications. Tetrazine-APS was synthesized, purified, and charac- conc: 5 mg mL−1), the resulting solution was added 3 µL of the
terized according to previous reported procedures.[6]
TCO-PEG12-DBCO stock solution (50 × 10−3 m in DMF), and the reac-
Synthesis of CPD-ASO-Dox-qMSN: As shown in Figure S2a in tion was stirred overnight, followed by centrifugation and washing
the Supporting Information, 3-aminopropyl-functionalized MSNs with DMF and DI water (2 times), then resuspension in 1.0 mL PBS
were first synthesized according to previously reported protocols.[6] to give DBCO-qMSN (1.0 mg mL−1). A portion of the MSN solution
Briefly, CTAB (0.1 g) was first dissolved in ultrapure water (48 mL). (50 µL) was next added 6 µL of the capping ASO stock solution
NaOH (2.0 m, 0.35 mL) was added to the solution and stirred for (100 × 10−6 m) and the resulting mixture was incubated at 37 °C
15 min, and then the mixture was heated to 80 °C. TEOS (0.5 mL) for 2 h. Upon centrifugation, washing with water (3 times) and
was added dropwise into the above solution with constant stirring resuspension in 50 µL of PBS (final MSN conc: 1.0 mg mL−1), the
and APTES (0.1 mL) was added 5 min later. Upon further stirring solution was added 5 µL of the N3-RGD stock solution (50 × 10−3
m
at 80 °C for 2 h, the resulting nanoparticles were filtered, washed in H2O) followed by incubation at room temperature for 4 h. Upon
with methanol, and deionized (DI) water, and freeze-dried over- centrifugation, washing with DI water (2 times) and resuspension
night. The dried 3-aminopropyl-functionalized MSNs with CTAB in PBS (50 µL), the resulting RGD-ASO-qMSN stock was obtained
were used for further modification as follows. The MSNs with CTAB (1.0 mg mL−1).
(1.0 mg) were dispersed in 5 mL dry toluene, followed by addition
Fluorescence Quenching and Drug-Loading Efficiency of qMSN:
of 50 µL (or other amounts as indicated in Figure S4 in the Sup- To determine the fluorescence quenching efficiency of ASO-qMSN
porting Information) of BHQ-2-APS and/or 50 µL of N3-APS. The prepared from modification with different amounts of BHQ-2-APS,
mixture was stirred at 50 °C in a nitrogen atmosphere for 6 h. The 20 µL of the TMR-ASO-capped nanoparticles (1 mg mL−1 in PBS)
nanoparticles were collected by centrifugation and washed with were transferred to each of two identical 1.5 mL centrifuge tubes
methanol and DI water, then freeze-dried overnight. The CTAB (A and B). Tube A was added 2 µL of synthetic survivin mRNA (1 ×
template from the obtained BHQ-MSN (qMSN; with or without N3 10−3 m; to prevent the heat-released TMR-ASO from binding back
group) was subsequently removed by dispersing the nanoparticles to qMSN surface), and the sample was heated at 95 °C for 10 min
in methanol (5 mL) containing concentrated HCl (37%, 0.3 mL). followed by dilution with 400 µL of PBS and fluorescence measure-
The resulting solution was stirred at room temperature for 24 h, ment on a Horiba FluoroMax-4 fluorometer (λex = 545 nm; λem
=
followed by centrifugation and washing with ethanol and DI water, 560–700 nm with max λem = 580 nm). The resulting fluorescence
giving qMSN without CTAB. Next, TCO surface modification was reading (measured at λem = 580 nm) from this sample was used
done by adding to the qMSN (1 mg dispersed in 0.2 mL PBS) 3 µL as Ifull release (100% TMR-ASO release). Tube B was directly added
of 50 × 10−3 m TCO-PEG12-DBCO stock solution (dissolved in DMF), 400 mL of PBS (no target and no heating) followed by fluorescence
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measurement to give Icontrol (0% TMR-ASO release). The QE of λem = 558 nm). Results are shown in Figure 3 and in Figures S5b–d
qMSN was calculated as follows and shown in Figures S4a,b in the in the Supporting Information.
Supporting Information
Cell Culture: HeLa, MCF-7, A549, A431, PC3, HepG 2, HEK
293T cell lines were cultured in DMEM cell culture medium
supplemented with 10% fetal bovine serum (Hyclone) and 2%
penicillin/streptomycin (PAN-Biotech) and maintained at 37 °C
in a 5% CO2 atmosphere. MCF-10A cells were cultured in mam-
mary epithelial cell growth medium (Lonza) supplemented with
100 ng mL−1 cholera toxin (Sigma Aldrich), and maintained at
37 °C in a 5% CO2 atmosphere.
Live-Cell Imaging: For CLSM imaging, cells were seeded in
4-well glass-bottom dishes (Greiner Bio-One) and grown until
50% confluency. After medium removal, cells were incubated with
10 µg mL−1 of different qMSNs (in fresh cell medium) for different
periods of incubation time. After that, cells were washed with PBS
Icontrol
Ifull release
QE% =
×100%
The Dox loading efficiency was determined, as previously
described,[6] by measuring the fluorescence spectra of the super-
natant after loading with stock solutions containing different Dox
concentrations, and comparing them with the standard calibration
curve (Figure S4d, Supporting Information insert; λex = 488 nm,
λem = 558 nm). The optimized loading amount of Dox to ASO-Dox-
qMSN was calculated to be 58.85 µmol g−1 (Figure S4d, Supporting
Information). The loading efficiency of YM155 was similarly calcu-
lated to be 72.34 µmol g−1, from a standard curve obtained from
YM155 stocks by measuring the UV–vis spectra (λabs = 348 nm).
The amount of BHQ-2 modified on the qMSN surface was similarly
determined with a standard curve generated from BHQ-2 stock
solutions (λabs = 579 nm). The amount of ASO capping on the
qMSN was done as previously described.[6] Briefly, by monitoring
the fluorescence intensity of ASO oligos at 580 nm (λex = 545 nm)
in the supernatant and comparing it against a standard curve
generated from ASO stock solutions (Figure S4c, Supporting Infor-
mation), the ASO capping amount for CPD-ASO-Dox-qMSN was
determined to be 18.46 µmol g−1. The density of ASO oligos on the
qMSNs surface was calculated as follows: d = c × V/m, where the
units of c, V, m, and d are in nmol mL−1, mL, mg, and nmol mg−1,
respectively.
Target Detection with Synthetic Survivin mRNA: 10 µL of CPD-
ASO-qMSN (1.0 mg mL−1 in PBS) were mixed with 85 µL of PBS
containing different concentrations of synthetic Survivin mRNA or
a control oligo, and the resulting mixtures were incubated at 37 °C
for 2 h and diluted with 300 µL of PBS (giving a final volume of
400 µL in each sample), followed by fluorescence measurements
(λex = 545 nm, λem = 580 nm). Time-dependent release experi-
ments were similarly done (0.1 mg mL−1 qMSN). Results are shown
in Figure 2 in the main text.
buffer twice, and then stained by Hoechst 33342 (VHoechst:VDMEM
=
1:30 000). The treated cells were analyzed on a Leica TCS SP5
X confocal microscope equipped with a 63× water immersion
objective (λex = 405 nm, λem = 430–460 nm for Hoechst 33342;
λex = 488 nm, λem = 565–620 nm for Dox and λex = 545 nm,
λem = 565–620 nm for TMR). For 3D imaging, cells were seeded in
4-well glass-bottom dishes (Greiner Bio-One) and grown until 50%
confluency. Upon removing medium, cells were incubated with
10 µg mL−1 of the different qMSNs for different lengths of incu-
bation time, then washed with PBS buffer (2 times) and nuclei-
stained by Hoechst 33342. A FV1000-X81 confocal microscope
(Olympus) equipped with a 60× water immersion objective was
used to image the treated cells (λex = 405 nm, λem = 415–475 nm
for Hoechst 33342; λex = 488 nm, λem = 500–550 nm for Dox, and
λex = 561 nm, λem = 580–650 nm for TMR). Results are shown
in Figure S8 in the Supporting Information and in the main text
(Figure 4).
High-Content Screening: For imaging-based, HCS experi-
ments, different cell lines were seeded in 96-well plates as pre-
viously described.[1] When cells reached 60% confluency, they
were incubated with 10 µg mL−1 of CPD-ASO-qMSN for 4 h. Subse-
quently, cells were washed with PBS (2 times) and nuclei-stained
by Hoechst 33342 as earlier described. Images were acquired
by employing In-Cell Analyzer automated fluorescence imaging
system (GE Healthcare) equipped with a 20× objective. Four
images were randomly acquired from each well with a laser-based
Target Detection with Total RNA from HeLa Cell Lysates: HeLa
cells were first seeded in 12-well dishes and incubated until 80%
confluency. Upon medium removal, the cells were washed with
PBS (2 times) followed by total RNA isolation using TRIzol Rea-
gent (Invitrogen) according to manufacturer’s protocols.[6] The
autofocus system under the same exposure conditions (λex
390 18 nm, λem = 432.5 48 nm for Hoechst 33342; λex = 542
=
27 nm, λem = 597 45 nm for TMR; λex = 542 27 nm for bright-
field). On average, around 300 cells were analyzed in each well.
All images were subsequently processed with In-Cell Developer
Toolbox software 1.9.2 by following the vendor’s protocols.[20a]
Both the nucleus and CPD-ASO-qMSN were analyzed, and the
nucleus was measured by counts while TMR fluorescence released
from CPD-ASO-qMSN was measured by the sum of TMR fluores-
cence intensity in each cell. The average intensity of released TMR
fluorescence from CPD-ASO-qMSN in each cell was calculated
by using the sum released TMR fluorescence/Count of Hoechst.
Results are shown in Figure S10 in the Supporting Information.
Flow Cytometry: For such experiments, 10 µg mL−1 of CPD-
ASO-qMSN (prepared in fresh cell medium) were incubated
with different cells (grown to 60% confluency) in 24-well plates
at 37 °C for 4 h. The cells were washed with cold PBS buffer
(2 times) before being detached by treatment with 200 µL of
isolated RNA was quantified (UV260/280
measurement) and
nm
used immediately in the following experiments. Briefly, different
amounts of these total RNA isolates were added to 10 µL of ASO-
qMSN (1.0 mg mL−1 in PBS), and the final volume of the mixture
was adjusted 100 µL with PBS. The mixture was next incubated at
37 °C for 2 h, followed by fluorescence measurements (Figure S5a,
Supporting Information).
Dox Controlled-Release Experiments: Typical in vitro controlled
release experiments of Dox were performed in PBS or DMEM.
First, CPD-ASO-Dox-qMSN were dispersed in PBS/DMEM solu-
tion to a final concentration of 0.1 mg mL−1. Subsequently, dif-
ferent amounts of synthetic survivin mRNA (or a control oligo; in
100 × 10−6 m stock) were added and the resulting mixtures were
incubated at 37 °C. Aliquots of the mixtures were taken peri-
odically, centrifuged (at 13,200 rpm) to collect the supernatant
whose fluorescence was subsequently measured (λex = 488 nm,
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0.25% trypsin-EDTA (ethylenediaminetetraacetic acid) at 37 °C Reverse Transcription Kit (Applied Biosystems) was used to reverse
for 2 min, then collected by centrifugation (2 min at 1000 rpm) at transcribe RNA to cDNA (25 °C, 10 min; 37 °C, 120 min; 85 °C,
4 °C. Upon further washing with 200 µL cold PBS (2 times), the 5 min; 4 °C, ∞). The qPCR was conducted with a TaqMan Uni-
cells were resuspended in 500 µL PBS, followed by analysis on a versal Master Mix II (no UNG) on a Bio-Rad MyiQ RT-PCR thermo
BD Accuri C6 cell analyzer (min. 10 000 cells were counted). Cell cycler (95 °C, 10 min; followed by 40 cycles of 95 °C, 15 s; 60 °C,
lines without qMSN-treatments were run concurrently as negative 60 s). The corresponding primers used in this experiment were pur-
controls. Results are shown in Figures 4 and 5.
chased from Life Technologies (Hs04194392_s1, control primers:
Apoptosis, Cell Viability, and Western Blotting: Cells were Hs03929097_g1). The relative expression of survivin mRNA
seeded and grown to 70–80% confluency in 12-well plates, was quantified by normalization against an endogenous con-
then incubated with different qMSNs (50 µg mL−1) at 37 °C for trol of GAPDH mRNA by using the 2−ΔCT method,[6] in which ΔCT =
24 h. Cells without treatment with qMSNs were concurrently run CTsurvivin − CTGAPDH. All qPCR reactions were performed in triplicate.
as negative controls. Subsequently, cells were washed with cold Results are shown in Figures S9, S10, and S14 in the Supporting
PBS (2 times) and detached with 0.25% trypsin-EDTA, followed by Information.
centrifugation and washing with cold PBS. Propidium iodide (PI)
and Annexin V-FITC staining was next carried out by using a FITC
Annexin V Apoptosis Detection Kit (BD Pharmingen, #556547)
according to the manufacturer’s guidelines and previously pub-
lished protocols.[26a] Briefly, upon washing, cells were resuspended
in 1× binding buffer at a concentration of 1 × 106 cell mL−1. 100 µL
Supporting Information
the above cells were transferred to a 1.5 mL centrifuge tube and
then co-stained with 5 µL of FITC Annexin V and PI each, followed
by addition of 400 µL 1 × binding buffer. Subsequently, the treated
cells were analyzed on a BD Accuri C6 cell analyzer (min. 10 000
cells were counted). Cell viability was performed by using XTT col-
orimetric cell proliferation kit (Roche) following manufacturer’s
guidelines and previously published protocols.[33] Briefly, HeLa
cells were seeded in a 96-well plate and grown to 50–60% conflu-
ency. Cells were then treated with different qMSNs (50 µg mL−1)
in fresh DMEM medium. Cells without qMSN treatment were done
concurrently as control samples. After 24 h of incubation, 50 µL of
the XTT reagent solutions were added into each well followed by
incubation for another 2 h. Then proliferation absorbance of the
solution was assayed by using a Bio-Rad 680 microplate reader (at
450 nm, with reference at 650 nm). Data represented the average
(s.d.) of triplicates. For WB, HeLa cells were first seeded in 12-well
plates and grown to 70–80% confluency, then added different
qMSNs (50 µg mL−1 in DMEM medium) followed by incubation at
37 °C for 24 h. Cells without any treatment were used as nega-
tive controls. The cells were next washed with cold PBS (3 times),
collected and the resulting cell pellets were lysed in the Laemmli
buffer (62.5 × 10−3 m Tris-HCl, pH 6.8, 20% glycerol, 2% SDS,
2 × 10−3 m DTT, Halt phosphatase inhibitor and Roche proteinase
inhibitor cocktail) by boiling at 95 °C for 10 min. Protein concentra-
tion was determined by using the Bradford Protein Assay (Bio-Rad,
#162-0177) as previously described.[2] Subsequently, WB analysis
was performed by using the appropriate primary and secondary
antibodies according to previously reported protocols.[6]
Downregulation and qPCR: For YM155-induced downregula-
tion experiment of survivin mRNA in HeLa cells, the cells were first
seeded in 24-well plates for 12 h, then incubated with different
amounts of a YM155 stock solution (100 × 10−6 m in DMSO) at
37 °C for 48 h. The intracellular expression levels of survivin mRNA
were determined by real-time quantitative PCR (qPCR) analysis.
Next, cells (at 80% confluency) were incubated with different
qMSNs (50 µg mL−1) at 37 °C for 24 h. Upon medium removal, RNA
isolation was performed using TRIzol Reagent (Invitrogen) following
the manufacturer’s protocols. The concentration of total RNA was
determined and adjusted to 10 ng µL−1 by using a Nanodrop
spectrometer (UV260/280 nm). Next, a TaqMan High-Capacity cDNA
Supporting Information is available from the Wiley Online Library
or from the author.
Acknowledgements
Funding was provided by National Medical Research Council
(CBRG/0038/2013), Ministry of Education (MOE2013-T2-1-048)
of the Singapore government and Natural Science Foundation of
China (51403065, sklssm201730). The authors thank Yan Tong
(NUS) for assistance with 3D images, and Prof. Yong Zhang (NUS)
for use of Nano-ZS90 instrument.
Conflict of Interest
The authors declare no conflict of interest.
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Received: February 20, 2017
Revised: March 17, 2017
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