A selective release system based on dual-drug-loaded mesoporous silica for nanoparticle-assisted combination therapy

Article abstract of DOI:10.1002/chem.201402334

A selective release system was demonstrated with a dual-cargo loaded MSNs. When stimulated by different signals (UV or H⁺), this system could selectively release different kinds of cargoes individually. Furthermore, this system has been used to provide a combination of chemotherapy and biotherapy for cancer treatment. This controlled release system could be an important step in the development of more effective and sophisticated nanomedicine and nanodevices, due to the possibility of selective release of a complex multi-drug. Combined cargo combats cancer: A selective release system was demonstrated with dual-cargo-loaded mesoporous silica nanoparticles (MSNs). When stimulated by different signals (UV or H⁺; see figure), this system could selectively release different kinds of cargoes in turn. In addition, this system has been used to provide a combination of chemotherapy and biotherapy for cancer treatment.

Full text of DOI:10.1002/chem.201402334

DOI: 10.1002/chem.201402334
Full Paper
&
Drug Delivery
A Selective Release System Based on Dual-Drug-Loaded
Mesoporous Silica for Nanoparticle-Assisted Combination Therapy
Wenqian Wang, Yongqiang Wen,* Liping Xu, Hongwu Du, Yabin Zhou, and Xueji Zhang*[a]
Abstract: A selective release system was demonstrated with
a dual-cargo loaded MSNs. When stimulated by different sig-
nals (UV or H+), this system could selectively release differ-
ent kinds of cargoes individually. Furthermore, this system
has been used to provide a combination of chemotherapy
and biotherapy for cancer treatment. This controlled release
system could be an important step in the development of
more effective and sophisticated nanomedicine and nanode-
vices, due to the possibility of selective release of a complex
multi-drug.
goes.[8] In spite of the substantial progress, most of these con-
trolled-release systems contain only one kind of cargo. Recent-
ly, some controlled-release systems loaded with multiple car-
goes had been designed. However, these systems were limited
to release the multiple cargoes simultaneously or release them
in sequence in different size ranges.[9] It still remains a grand
challenge to develop a multi-cargo-loaded system that can
control the release of each cargo independently at will.
Introduction
The controlled release system, which has been widely used in
drugs delivery, cell imaging, disease diagnosis, catalysis, and
environmental protection, has gained much attention in the
past couple of decades.[1–4] In particular, it shows important ap-
plications in the treatment of severe diseases. Cancer is one of
the most serious diseases that threaten human beings today.
So far, multiple cancer treatment technologies, such as surgical
resection, radiotherapy, chemotherapy, thermotherapy, biother-
apy, and gene therapy, have been widely used to treat cancer;
however, they all have their own limitations. For example, che-
motherapy, as the most common method for cancer treatment,
has the problem of cancer chemoresistance, which leads to
most of the failed cases in cancer therapy.[5] These challenges
have driven researchers and clinicians to investigate clever and
elegant approaches to overcome these limitations. The combi-
nation of multiple therapy technologies/drugs has been re-
garded as one of the most promising treatment strategies,
which can reduce the side effects, suppress drug resistance,
and strengthen the antitumor effect through distinct mecha-
nisms of action.[5,6]
In this paper, a selectively controlled-release system was
demonstrated through
a dual-cargo-loaded MSNs system
(Scheme 1). In this system, the supramolecular pseudorotax-
anes were used to trap two kinds of cargoes into the nano-
pores of MSNs individually. These nanopores were radially ori-
ented and unconnected between each other. Under the stimuli
of different signals, UV or H+, the dual cargoes could be selec-
tively released from the MSNs separately. In the neutral solu-
tion, only one cargo could be released from the MSNs under
the irradiation of UV; and whether UV or not, only another
As one of the most extensively studied controlled-release
platforms, the mesoporous silica nanoparticle (MSN) is espe-
cially attractive for its large surface exteriors, porous interiors,
biocompatibility and the ability to chemically modify its sur-
face.[7] To date, many delicate intelligent switches had been im-
plemented by other researchers and ourselves to trigger the
opening of the pores and the release of encapsulated car-
[a] Dr. W. Wang, Prof. Y. Wen, Prof. L. Xu, Prof. H. Du, Y. Zhou, Prof. X. Zhang
Research Center for Bioengineering &
Sensing Technology, School of Chemistry &
Biological Engineering, University of Science and
Technology Beijing, Beijing 100083 (P.R. China)
E-mail: wyq_wen@iccas.ac.cn
Scheme 1. The release process of the dual-dye-loaded MSNs. The dual dyes
were loaded into the MSNs separately by pH-controlled nanogates and UV-
controlled nanovalves. This system can selectively release Eosin Yellowish
(EY) upon UV irradiation (at pH 7.0) and Rhodamine B (RhB) at pH 3.5.
zhangxueji@ustb.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402334.
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cargo could be released from the MSNs upon the stimulus of
H+. In consideration of the low pH of intracellular lysosome
and nearly neutral pH of most extracellular fluids, the selective
release system had been further applied to release two kinds
of antitumor agents in the intracellular and extracellular envi-
ronment of tumor cell, individually, which take effect on tumor
cells through chemotherapy and biotherapy, respectively.
Under the combination of two cancer treatment technologies,
the inhibitory rate of tumor cells A549 was evidently increased
with respect to a single treatment. This research has made an
important progress in MSNs-based intelligent controlled-re-
lease system. The selective controlled-release system would
allow more accurate and controllable release for a sophisticated
drug delivery, and could have the potential to treat human dis-
eases effectively with combination therapies and diagnosis.
Results and Discussion
As shown in Figure 1, MSNs were obtained by using the modi-
fied base-catalyzed sol–gel method with an average diameter
of 350 nm.[10] The mesoporous channel could be clearly ob-
served by transmission electron microscopy. N2 sorption analy-
sis of the MSN exhibited a type IV isotherm with a total surface
area (Brunauer–Emmett–Teller, BET) of 1766.59 m2 gÀ1 and an
average pore diameter of 4.4 nm. The nanoparticles also
showed typical XRD patterns of MCM-41 type hexagonal meso-
porous silica with a lattice spacing of about 4.3 nm.
Figure 1. a) SEM: The average diameter of MSN was 350 nm. b) TEM: The
mesoporous channels were clearly observed. The scale bar corresponds to
50 nm. c) N2 sorption isotherm. Inset: pore size distribution. d) XRD.
functional MSNs (MS-PhBPDB; Scheme 2 and Figure S2, the
Supporting Information). In the UV/Vis spectrum of MS-
PhBPDB (Figure 2), the absorption peak at 250 nm was attrib-
uted to the benzene group, and the absorption peak at
340 nm was produced by azobenzene group.
As has been frequently reported in literature,[11] a-cyclodex-
trins (a-CD) have a strong propensity for binding with the Ph
group and a much weaker propensity with protonated Ph
group. And because of the much lower efficient photoisomeri-
zation of azobenzene unit in acidic solution,[12] b-cyclodextrins
(E)-4-((4-(Benzylcarbamoyl)phenyl)diazenyl) benzoic acid
(BPDB) was firstly synthesized by the dehydration between
azobenzene-4, 4’-dicarboxylic acid and benzylamine (Figure S1,
the Supporting Information).[11a] MSNs were treated with 3-ami-
nopropyltriethoxysilane (APTES) to get the amine-modified
MSNs (MS-NH2). Then, the MS-NH2 was modified with BPDB
and N-phenylaminopropyltrimethoxysilane (PhAPTMS) step by
step to afford the phenylamine group (Ph) and BPDB dual-
Scheme 2. Preparation of the dual-dye loaded MSNs. (1) APTES, BPDB; (2) PhAPTMS; (3) b-CD; (4) RhB; (5) a-CD; (6) UV light; (7) EY; (8) b-CD.
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Figure 3. Release behavior of dual-cargo-loaded MS-PhBPDB, without
capped cyclodextrins, at pH 7.0 without UV irradiation. This demonstrates
that the dual cargoes could be released from the MS-PhBPDB simultaneous-
ly without the CD capped in the aqueous solution (i: 0; ii: 0.5; iii: 1.0; iv:
1.5; v: 3.0; vi: 4.0 h).
Figure 2. The UV/Vis absorption spectra of the aqueous solution of different
functional MSNs (1 mgmLÀ1 MSNs; i: MS-PhBPDB; ii: MS-BPDB; iii: MS-Ph; iv:
MS; v: control). The absorption peak at 250 nm was attributed to the ben-
zene group, and the absorption peak at 340 nm was produced by the azo-
benzene group.
prevented from leaking from the MSNs at pH 7.0, and the two
kinds of dyes escaped from the nanopores simultaneously.
However, in the presence of cyclodextrins, none of the two
dyes were observed at the conditions of pH 7.0 without the ir-
radiation of UV. The flat baseline indicates that both of the two
dyes were not released in the neutral solution (Figure 4a and
4b). The results proved that the complexes of Ph/a-CD and
BPDB/b-CD had formed the stiff structure of pseudorotaxanes
to cap the pores of the MSNs in neutral solution. Hence, both
of the dyes could not be released from the nanopores.
(b-CD) can only reversibly bind with azobenzene unit through
the trans–cis photoisomerization of azobenzene unit under UV
irradiation in neutral conditions (Figure S4, the Supporting In-
formation). Therefore,
a supramolecular pseudorotaxanes
system containing the a-CD rings on PhAPTMS grafting a Ph
group can act as “gatekeepers” of the nanopores on the MSNs.
This system blocks the departure of cargo molecules at physio-
logical conditions, as well as another pseudorotaxanes assem-
bled by b-CD rings threading onto BPDB stalks containing an
azobenzene group. These two supramolecular structures, Ph/
a-CD and BPDB/b-CD, could be disassociated to unblock the
nanopores of MSNs by decreasing the pH value of external
medium and UV irradiation at pH 7.0, individually. In addition,
the binding affinity between a-CD and azobenzene group or
between b-CD and the Ph group is much lower to assemble
the stiff psudorotaxanes structures for capping the nanopores
on the MSNs (Figure S5 and S6, the Supporting Information).
For above all, the method of “close one, open another” was
employed to load Rhodamine B (RhB) with Ph/a-CD nano-
valves and Eosin Yellowish (EY) with BPDB/b-CD nanovalves
into the MS-PhBPDB, respectively (Scheme 2). In a typical ex-
periment, the pores modified by BPDB were firstly capped by
b-CD to stop the cargoes diffusing into these pores. With one
kind of the fluorescent molecules (RhB) loaded into the open
nanopores, these nanopores were locked by a-CD rings bind-
ing to Ph-containing stalks. Subsequently, the RhB-loaded
MSNs were irradiated by UV light to disassociate the b-CD
rings from the BPDB stalks and open the nanopores that were
capped by b-CD. Then, the other kind of fluorescent molecules,
EY, were trapped into these pores by b-CD rings threading
onto the BPDB stalks. Fluorescence intensity at 540 (EY) and
575 nm (RhB) was used to monitor the signals of released
cargo molecules from the dual-dye loaded MSNs.
The dual-dye-loaded MSNs were then irradiated by UV light,
while the external medium was maintained at pH 7.0. The
result showed that a rapid increase in the emission intensity
Figure 4. The release behavior of dual-dye-loaded MSNs controlled by single
stimulus. a) UV-Light dependence of the release profiles of EY and b) pH de-
pendence of the release profiles of RhB from the MSNs system. c, d) Fluores-
cence spectra of dual-dye loaded MSNs before (i) and after (ii) irradiating
with UV light at pH 7.0 or lowering the pH to 3.5 for 4 h.
As illustrated in Figure 3, when the dual-dye loaded MSNs
were tested without a cyclodextrin cap, the dyes could not be
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around 540 nm occurred, whereas another emission signal
(575 nm) was not observed at all. The results indicated that
the EY was released from the nanopores of the MSNs, but RhB
was still retained in the MSNs upon UV-light irradiation. This
consequence could be attributed to the conformational
changes of the azobenzene unit of BPDB from the more stable
trans to the less stable cis configuration. The isomerization of
the azobenzene unit led to the dissociation of pseudorotax-
anes (BPDB/b-CD). The b-CD rings were unthreaded from the
BPDB stalks and they unblocked the nanopores sealed by the
BPDB/b-CD complex. Under the same conditions, the pseudo-
rotaxanes of Ph/a-CD were still stable and stopped the RhB es-
caping from the MSNs. Therefore, only EY molecules were re-
leased from the nanopores of MSNs (Figure 4a and c).
duced the rate of photoisomerization to inhibit the b-CD rings
unthreaded from the BPDB stalks under UV irradiation. There-
fore, EY was still stored in the nanopores of MSNs and no re-
lease of EY was observed under these conditions.
As a preliminary application research, the tests of cell viabili-
ty were further carried out to evaluate the combination thera-
py efficiency of the selective release system on A549 cell lines.
By using a combination of chemotherapy and biotherapy, dif-
ferent kinds of antitumor agents could take effect at individual
active sites, at specific times and in a different dosage. The
bio-antitumor agents can bind the receptor on the surface of
the tumor cell to induce tumor cell apoptosis in extracellular
environment, whereas most of the chemotherapy drugs take
effect in an intracellular environment.[13] In our experiment, oc-
treotide acetate (OCT) and epirubicin (EPB) were used as the
model biotherapy and chemotherapy antitumor agents to load
into the MS-PhBPDB step by step. OCT, a kind of somatostatin
analogue, has antitumor activities mediated through somato-
statin receptors over-expressed on the surface of tumor cells in
extracellular environment; whereas EPB could embed into two
nucleic acid bases to kill the tumor cells through interfering
the transcription process and inhibiting the synthesis of DNA
and RNA after entering the tumor cells. For the reason of the
low pH of intracellular lysosome and nearly neutral pH of most
extracellular fluids, the UV-responsive nanogates on the MS-
PhBPDB could control the drugs to release under the stimulus
of UV irradiation only in extracellular environment but not in
an intracellular environment; and, the pH-responsive nano-
gates could control the drugs to release only in intracellular
environment after the MS-PhBPDB entered into the cell
through endocytosis.
When the pH value of the solution was adjusted to 3.5 and
no UV light was provided, an obvious increase in the emission
intensity only around 575 nm occurred, indicating that the RhB
escaped from the nanopores of the MSNs upon lowering the
pH, whereas the EY was still sustained in the MSNs. The addi-
tion of H+ could lead to the protonation of Ph group, and the
binding affinity between a-CD and the Ph group would be di-
minished, causing the dissociation of the a-CD rings from the
PhAPTMS stalks. The a-CD rings unblocked the nanopores
modified by the PhAPTMS and allowed the RhB to escape into
solution. Under the same conditions, the b-CD rings did not
unthread from azobenzene-containing BPDB stalks in the acid
solution (Figure 4b and 4d). The fluorescence spectra of the
release system (Figure 4c and 4d) after UV irradiation and H+
addition showed dramatically different characteristic peaks,
which proved that the dual-dye-loaded MSNs can selectively
release the EY or RhB molecules in response to different exter-
nal stimuli.
In our experiment, EPB and OCT were loaded into the MS-
PhBPDB by pH-controlled and UV-controlled nanovalves, re-
spectively. A549 cells were incubated with this dual-drug-
loaded MS-PhBPDB for 4 h, and then washed twice with PBS
buffer before adding fresh growth medium for further incuba-
tion 24 h. As shown in Figure 6aii, the viability of the A549
cells decreased to 42.83% compared with the control (Fig-
ure 6ai). Considering that the lower pH of tumor interior tis-
sues, endosomes and lysosomes, the result could be attributed
to the release of EPB from MS-PhBPDB, which entered into the
tumor cells through endocytosis. In addition, the mixture of
cell cultures and MS-PhBPDB was irradiated by UV for 15 min
and incubated in dark. After 24 h, the cell cultures were sepa-
rated from the MS-PhBPDB and incubated with the tumor cells
for another 24 h. As shown in Figure 6aiii, the viability of A549
cells decreased to 57.07% due to the apoptosis of tumor cells
induced by the release of OCT. Finally, the dual-drug-loaded
MS-PhBPDB was incubated with A549 cells and irradiated im-
mediately with UV for 15 min. The viability of the cells de-
creased significantly to 26.55% after 24 h (Figure 6aiv, Fig-
ure 6b), which could be attributed to the combination therapy
of OCT and EPB at the extracellular and intracellular environ-
ment. In this case, the UV-controlled nanogates were firstly
opened to release OCT before the particles entering into the
cells. Then, EPB was sequentially released from the MS-PhBPDB
When the pH of the solution was adjusted to 3.5, the MSNs
were simultaneously irradiated by UV light. Interestingly, the
dual dyes, EY and RhB, did not release from the nanopores at
the same time. As shown in the fluorescence spectra, we can
only find the signal of RhB in the solution (Figure 5). In other
words, whether irradiating UV light or not, the nanopores
modified by BPDB stalks would be capped by pseudorotaxanes
of BPDB/b-CD constantly in acid solution. This consequence
may be attributed to the presence of the nitrogen atoms on
the BPDB stalks.[12] In acid solution, the protonation of the ben-
zylamine and two nitrogen atoms of the azobenzene group re-
Figure 5. The release behavior of dual-dye-loaded MSNs controlled by dual
stimuli. a) The release profiles of the MSNs upon UV irradiation at pH 3.5.
b) Fluorescence spectra of dual-dye-loaded MSNs before (i) and after (ii) irra-
diating with UV light in pH 3.5 for 4 h.
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lease allowed more accurate and controllable drug
delivery and high efficient therapy. The approaches
of selectively controlled release and combined thera-
py could be expected to be further extended to
more practical system, for example, the release
system activated by near-infrared (NIR), cancer bio-
markers, and so on, and it could also be applied to
the multiple combination therapy with other treat-
ment methods.
Experimental Section
Materials
Tetraethoxysilane (TEOS, 99.9%), n-cetyltrimethylammo-
nium bromide (CTAB, ꢀ99%), 3-aminopropyltriethoxysi-
lane (APTES, 99%), N-phenylaminopropyltrimethoxysi-
lane (PhAPTMS), Rhodamine B, Eosin Yellowish, azoben-
zene-4, 4’-dicarboxylic acid and 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide (MTT, ꢀ98%) were
purchased from Sigma Company. Benzylamine, thionyl
chloride, pyridine, sodium hydroxide, disodium hydro-
gen phosphate dodecahydrate, dimethyl sulfoxide, and
citric acid were purchased from Sinopharm Chemical
Reagent Co. Ltd. Octreotide acetate (OCT) and epirubi-
Figure 6. a) Viability test of A549 cells with dual-drug-loaded MS-PhBPDB (50 mgmLÀ1
after 24 h incubation, in which was loaded EPB and OCT by pH-controlled nanogates
and UV-controlled nanovalves, respectively (P<0.01). b) The images of A549 cells under
the combination therapy of OCT and EPB. The scale bar corresponds to 10 mm.
)
system after the MS-PhBPDB had been taken-up into the A549
cin (EPB) were purchased from Chengdu Kaijie Biopharm Co. Ltd.
and Pfizer. RPMI Medium 1640 and fetal bovine serum (FBS) were
purchased from life technologies. All buffers were prepared with
ultra-pure MilliQ water (resistance>18 MW cmÀ1).
cells.
In a contrast experiment, OCT and EPB were loaded into the
MS-PhBPDB by pH-controlled and UV-controlled nanovalves,
respectively. After the MS-PhBPDB entered into the cells, the vi-
ability of A549 did not obviously decrease for the OCT releas-
ing at the intracellular environment, indicating that was an im-
proper delivery site for OCT (A549=92.07%, Figure S11ii and
S13A, the Supporting Information). Meanwhile, EPB was not re-
leased after the MS-PhBPDB entered into the A549 under UV ir-
radiation; it can only be released when the MS-PhBPDB is lo-
cated at the neutral environment (Figure S11iii, iv, and Fig-
ure S12B, the Supporting Information).
Instruments
Scanning electron microscopy (SEM) was performed with a JEOL-
6700FE instrument. Transmission electron microscopy (TEM)
images were obtained by using a TecnaiG2 F20 electron micro-
scope. Powder X-ray diffraction (XRD) patterns were collected
using a Rigaku D/max 2500 equipped with CuKa radiation. UV/Vis
spectra were collected using a Shimadzu U-1800 spectrophotome-
ter. All fluorescence spectra were recorded on a Hitachi F-4500 FL
Spectrophotometer in PBS buffer. Thermal stability measurements
were collected on DTG-60 (Shimadzu Instruments) between 100
and 5508C with a heating rate of 108CminÀ1. N2 adsorption-de-
sorption isotherms were obtained at 77 K on a Micromeritics
ASAP2020 automated sorption analyzer. The BET model was ap-
plied to evaluate the specific surface areas. Pore size and pore
volume were determined from the adsorption data by the BJH
method. 1H NMR spectra (400 MHz) were recorded on a Bruker
DPX 400 MHz spectrometer in CDCl3.
The cell experiments indicated that the UV-responsive nano-
gates could control the release of bio-antitumor agent, which
only takes effect in the extracellular environment of the tumor;
whereas the pH-responsive nanogates could control the che-
motherapy drugs to be released only in intracellular environ-
ment of the tumor. These results further proved that a high an-
titumor efficiency could only be realized when specific antitu-
mor agents were delivered and released, and only played an
active role at specific situations. Therefore, a precise, controlla-
ble, multidrug delivery system was pivotal for enhancing anti-
tumor efficiency and reducing the side effects.
Syntheses
BPDB: Azobenzene-4, 4’-dicarboxylic acid (0.20 g, 0.74 mmol) was
stirred with thionyl chloride (0.19 g, 1.6 mmol) and pyridine (0.16 g,
2.0 mmol) in CH2Cl2 for 1 h followed by addition of benzylamine
(0.08 g, 0.74 mmol). The mixture was stirred at room temperature
until complete disappearance of starting material as verified by
TLC (ꢁ2–3 h). After removal of the solvent, the residue was puri-
fied by column chromatography (CHCl3/MeOH 10:1) to afford pure
BPDB as a yellow oil (0.20 g, 74%). H NMR (400 MHz, CDCl3, 258C,
TMS): d=3.96 (s, 2H, CH2), 7.40–7.26 (m, 6H), 7.99--7.97 (t, 6H),
8.20 (d, 1H), 8.22 ppm (d, 1H);
Conclusion
We report here an intelligent MSNs-based multidrug selective
release system. This system could selectively release different
cargoes under the stimuli of different signals. This system was
further applied to the combination of chemotherapy and bio-
therapy of cancer through loading and releasing two kinds of
antitumor agents at intracellular and extracellular environment
of the tumor cell, respectively. The selectively controlled re-
1
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MSN: N-Cetyltrimethylammonium bromide (CTAB, 0.5 g) was mixed
with water (42 mL), ethanol (18 mL) and solution of sodium hy-
droxide (0.6 mL, 2 molLÀ1) under stirring for 30 min. Then, tetrae-
thoxysilane (TEOS, 2.8 mL) was once added to the mixture, which
was stirred at room temperature for 8 h. The resulting MSNs were
separated by centrifugation, washed several times with water and
ethanol, and dried in the oven (608C) overnight. Removal of tem-
plate was achieved by solvent extraction: MSNs (1.0 g) were sus-
pended in ethanol (100 mL) and 37% HCl (2 mL), and the mixture
was heated under reflux for 24 h. The solvent-extracted particles
were washed extensively with ethanol and collected through cen-
trifugation.
The studies of inhibition rate of A549 cells with OCT and
EPB
The A549 cells were placed on 96 wells cell culture clusters at
a density of 1ꢁ104 cells per well and cultured in 5% CO2 at 378C
for 12 h. Then, a different density of OCT and EPB were added to
the media, respectively, and the A549 cells were incubated in 5%
CO2 at 378C for 24 h. Cell viability was determined by the standard
MTT assay.
The biocompatibility studies of MS-PhBPDB and UV light
MS-PhBPDB: BPDB and phenylamine group-functionalization of
the silica surface was performed by suspending the MSNs (100 mg)
in a solution of 3-aminopropyltriethoxysilane (11 mL, 0.05 mmol) in
dry toluene (10 mL) for 30 min. The toluene was then removed by
centrifugation. The nanoparticles were washed with toluene, etha-
nol, and dried under vacuum, to give MS-NH2 (89.10 mg, 77.23%).
BPDB (17.97 mg, 0.05 mmol) was dissolved in dried toluene (5 mL).
The solution was added to flask containing 3-aminopropyltriethox-
ysilane-coated MSNs (50 mg). The solution was mixed and sonicat-
ed to disperse nanoparticles. The solution was then allowed to stir
under room temperature for 24 h, to give MS-BPDB (43.43 mg,
68.00%). MS-BPDB (50 mg) was treated with PhAPTMS (6 mL,
0.025 mmol) in dried toluene (10 mL) and allowed to heat at reflux
under N2 for 24 h, to give MS-PhBPDB (47.01 mg, 62.41%). The
nanoparticles were separated from solution by centrifugation and
washed with toluene and ethanol, in turn. The particles were dried
under vacuum overnight.
A549 cells were plated on 96 wells cell culture clusters at a density
of 1ꢁ104 cells per well and cultured in 5% CO2 at 378C for 12 h.
Then, MS-PhBPDB (100 mL, 50 mgmLÀ1) were added to the media
and irradiated by UV for 15 min, and the cells were incubated in
5% CO2 at 378C for 24 h. Cell viability was determined by the stan-
dard MTT assay.
Cell experiment
To test the release of the drugs in the cell line, A549 cells were
placed on 96 well cell culture clusters at a density of 1ꢁ104 cells
per well and cultured in 5% CO2 at 378C for 12 h. Before the load-
ing of the OCT and EPB, a solution of OCT (50 mgLÀ1) and EPB
(125 mmolLÀ1) had been prepared. Then, the MS-PhBPDB was di-
vided into two groups, and these two antitumor agents were
loaded in the same way as the loading of the dyes. Group I con-
tained trapped OCT with Ph/a-CD nanovalves and EPB with BPDB/
b-CD nanovalves into the MS-PhBPDB, respectively. Group II con-
tained trapped OCT with BPDB/b-CD nanovalves and EPB with Ph/
a-CD nanovalves into MS-PhBPDB, respectively. Subsequently, as-
sisted with ultrasonication, the two groups of dual-drug-loaded
Dual dyes loaded in the MS-PhBPDB: Firstly, b-CD (56.75 mg,
0.05 mmol) was added to an aqueous solution containing MS-
PhBPDB (50 mg) to cap the nanopores near the BPDB stalks on the
surface of nanoparticles. Then, the pH-controlled nanopores were
opened by dispersing the MSNs in acid solution (pH 3.5) and re-
covered by washing the nanoparticles with a neutral buffer solu-
tion. Secondly, the loading of RhB into MSNs was achieved by
soaking MS-PhBPDB (50 mg) in the an aqueous solution (10 mL)
containing RhB (0.8 mL, 0.1 mmolLÀ1) for about 24 h. Subsequently,
a-CD (48.64 mg, 0.05 mmol) was added to the solution and incu-
bated overnight to cap the nanopores controlled by pH. Next, the
RhB-loaded MSNs were irradiated by UV for 24 h to open the nano-
pores beside the azobenzene unit-containing stalks (washed the
MSNs in each 4 h). Finally, we used the way of loading RhB to load
the EY (0.4 mL, 0.1 mmolLÀ1) into the nanopores controlled by UV
and capped by b-CD again to obtain the dual-dye-loaded MS-
PhBPDB (49.2 mg, 59.58%).
MS-PhBPDB were dispersed in cell culture fluid (100 mL, 50 gmLÀ1
,
1640 medium+8% FBS), and added to the media, separately.
Next, the two groups were irradiated by UV for 15 min and incu-
bated in 5% CO2 at 378C without visual light. After 24 h, cell viabil-
ity was determined by the standard MTT assay.
Test for the extracellular and intracellular release of dual-
drug-loaded MS-PhBPDB
A549 cells were placed on 96 well cell culture clusters at a density
of 1ꢁ104 cells per well and cultured in 5% CO2 at 378C for 12 h.
The two groups MS-PhBPDB (100 mL, 50 mgmLÀ1) was added to the
media, then A549 cells were incubated with dual-drug-loaded MS-
PhBPDB for 4 h, and then washed with PBS twice before adding
fresh growth medium for further incubation 24 h. As a control ex-
periment, the two groups were irradiated by UV for 15 min after
adding fresh growth medium. Cell viability was determined by the
standard MTT assay. In addition, the two groups MS-PhBPDB
(50 mgmLÀ1) were also placed at the corner of the quartz cuvette,
then the cell culture fluid was added into the cuvette. After 24 h,
the cell culture fluid was incubated with cells for another 24 h. As
a control experiment, the mixture of cell culture fluid and dual-
drug-loaded MS-PhBPDB were irradiated with UV for 15 min and
placed in dark for 24 h. Then, the cell culture fluid was incubated
with cells for another 24 h. Cell viability was determined by the
standard MTT assay.
Testing the dye release: The release signal was investigated by
monitoring the fluorescence intensity of the release system. Dual-
dye-loaded MSNs (3 mg) were placed in a bottom corner of cuv-
ette, and the PBS solution (citric acid/disodium hydrogen phos-
phate) was then carefully filled to avoid disturbing the MSNs.
Under some stimuli, the dyes would be released into the solution.
Without stimuli, the dyes would not be released from the MSNs
and diffused into the solution. Then, the solution could be directly
monitored by fluorescence spectrophotometer to record the re-
lease of dyes. For activating the nanomachines, the pH of the solu-
tion was decreased or MSNs were irradiated with UV-light, respec-
tively. The amount of released guest molecules from the pores of
MSNs was monitored by the change of the fluorescent intensity at
540 and 575 nm.
Chem. Eur. J. 2014, 20, 7796 – 7802
www.chemeurj.org
7801
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Acknowledgements
The authors would like to thank the NSFC (21171019,
21073203, 51373023, 21275017, 21127007, 21103009), Beijing
Natural Science Foundation (2122038), and the Fundamental
Research Funds for the Central Universities and NCET-11–0584.
Keywords: combination therapy · drug delivery · mesoporous
materials · nanotechnology · selective release
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Received: February 24, 2014
Published online on May 23, 2014
Chem. Eur. J. 2014, 20, 7796 – 7802
www.chemeurj.org
7802
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim