Confined Singlet Oxygen in Mesoporous Silica Nanoparticles: Selective Photochemical Oxidation of Small Molecules in Living Cells

Article abstract of DOI:10.1021/acs.bioconjchem.6b00061

(Chemical Equation Presented) Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity. Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional compound.

Full text of DOI:10.1021/acs.bioconjchem.6b00061

Article
pubs.acs.org/bc
Confined Singlet Oxygen in Mesoporous Silica Nanoparticles:
Selective Photochemical Oxidation of Small Molecules in Living Cells
Takuma Nakamura,† Aoi Son,† Yui Umehara,† Takeo Ito,† Ryohsuke Kurihara,‡ Yuta Ikemura,‡
and Kazuhito Tanabe*,‡
†Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto,
615-8510, Japan
‡Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe,
Chuo-ku, Sagamihara, 252-5258, Japan
S
* Supporting Information
ABSTRACT: Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive
tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it
has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity.
Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles
to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet
oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This
intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also
confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle
phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We
demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional
compound.
proteins, makes it difficult to excite specified molecules, which
interact with particular ones, even under irradiation conditions
using selected wavelength. The second problem is the
generation of reactive oxygen species (ROS) including singlet
oxygen (1O2) that are formed from interactions between triplet-
excited-state molecules and molecular oxygen in the ground
state.10−13 ROS have been used for photodynamic therapy
(PDT), but ROS oxidizes biomolecules widely to damage living
cells and tissue. Therefore, ROS potentially cause serious side
effects as well as beneficial effects. These two barriers have
limited the broad application of photochemical reactions to
biological experiments designed to understand and regulate the
functions of living cells and organisms.
INTRODUCTION
■
Noninvasive remote control of cellular functions has attracted
considerable attention.1−4 Since the identification of cellular
functions is directly linked to the comprehension of life
phenomenon, drug design, and medical treatment, there are
increasing demands for the development of systems to regulate
their functions. In the past, various attempts have been made to
manipulate cellular functions using external stimuli. One of the
most valuable technologies for remote control of cellular
functions is the application of photoirradiation.5−9 Because of
its capability for spatial and temporal control of chemical
reactions, this technique has been widely applied to biological
experiments. Nevertheless, it has been difficult to implement
molecule-specific photoreactions in living cells for the following
reasons. First, the complexity of the chaotic environment of the
cells, which consists of numerous biomolecules ranging from
small molecules to biopolymers such as nucleic acids and
Received: February 1, 2016
Revised: March 23, 2016
© XXXX American Chemical Society
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Scheme 1. Synthesis of MSN-MB from MSN-CO2H
Figure 1. (A) Absorption spectra of MSN-MB (1.0 mg/mL, solid line) and MSN-CO2H (1.0 mg/mL, dashed line) in DMSO. (B) Fluorescence
spectra of MSN-MB (1.0 mg/mL (41 μM of MB units), solid line) and MB-NH2 (41 μM, dotted line) in DMSO (Ex. 630 nm). (C) TEM image of
MSN-MB.
In this study, we attempted to design a photochemical
molecular conversion system without any side effects using
mesoporous silica nanoparticles (MSN). The pores of the
MSN, which were functionalized by chemical modification,
provide a limited reaction field for small molecules in living
cells, meaning that a highly selective reaction can be
implemented. MSN have several favorable features: they are
easily fabricated, have a large surface area, have remarkable
transparency for a wide range of wavelengths, and provide
amorphous frameworks with a large number of silanol groups
for postmodification. Heretofore, due to these characteristics,
MSN have been widely applied as functional materials such as
catalysis14−17 for photoreaction or organic synthesis, drug
carriers,18−21 or sensing devices.22
RESULTS AND DISCUSSION
■
Fabrication and Characterization of MSN-MB. Initially,
we synthesized MSN-MB as shown in Scheme 1. Coupling of
carboxylic-functionalized MSN (MSN-CO2H) with MB de-
rivatives possessing a primary amino group (MB-NH2) gave the
desired nanoparticles, MSN-MB.
To confirm the incorporation of MB units to MSN, we
measured the absorption spectra. Monodisperse MSN-MB had
a similar spectrum to MB-NH2 between 300 and 900 nm, while
MSN-CO2H showed no absorption at these wavelengths
(Figure 1A). We also measured the fluorescence spectra of
MSN-MB (Figure 1B). Compared with MB-NH2, MSN-MB
showed a red-shifted fluorescence emission because of
congestion and stabilization of the MB units on the surface
of MSN. These results strongly indicate that MB units were
densely embedded in the inner pores. Transmission electron
microscopy images revealed that the pore structure was
maintained even after modification by MB (Figure 1C). The
size of MSN-MB and their pore size (φ) were estimated to be
200 60 and 3.5 nm, respectively. In MSN-MB, the MB units
were located mainly in the inner pores. The ratio of the inner
surface area to the total surface area of the nanoparticles was
calculated to be 96% (eq 1 and 2; see Experimental Section).
Because a large majority of the carboxylic acid substituents were
located in the inner pore, the modification by MB occurred
predominantly in these inner pores. The number of MB units
incorporated on the surface of MSN was estimated to be 41
nmol/mg.
These excellent properties of MSN prompted us to apply
MSN to photoregulation of biological functions. Our
hypothesis was as follows: (1) the pores of MSN can be used
for selection of small molecules from the complex mixture of
biomolecules in living cells; and (2) large biomolecules such as
proteins, sugars, and nucleic acids can be prevented by pore size
limitations from passing into the pore to approach the reaction
fields. Thus, modification of the pores of MSN allows selective
reactions on small molecules. We modified the pores of MSN
with methylene blue (MB) photosensitizer, which generates
1
singlet oxygen (1O2) upon photoirradiation. O2 generated in
the pore immediately oxidizes those molecules, that can enter
the pore. On the other hand, unreacted 1O2 becomes
inactivated before exiting the pore, and therefore MSN with
1
MB (MSN-MB) can greatly reduce the harmful effects of O2.
Cytotoxicity of MSN-MB upon Photoirradiation. An
attempt was made to demonstrate the photochemical function
of this system using the human lung carcinoma cell line A549.
Initially, we assessed the cytotoxic properties of monadelphous
MB-NH2 under photoirradiation conditions, before the
Eventually, we applied these MSN to control cellular functions.
By photoirradiation, we successfully converted anthracene
derivatives (preAQ) to anthraquinone (AQ), which caused
cell cycle arrest without any cytotoxicity.
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evaluation of properties of the MSN-MB. After administration
of MB-NH2 to A549 cells and incubation for 5 h to allow their
penetration into the cells, the medium was replaced with fresh
medium to avoid undesirable activation of drugs outside the
cells. We then exposed A549 cells to photoirradiation. As
shown in Figure 2, a negligible cytotoxic effect was observed in
Figure 3. (A) Visualization of singlet oxygen generated in the A549
cells by photoirradiation in the presence of MSN-MB. The
fluorescence intensity of SOSG (550 nm) in the cells, in which
singlet oxygen was generated by photoirradiation (0 or 1 h) in the
presence of MSN-MB (green) or MB-NH2 (yellow). The fluorescence
of SOSG in the absence of photosensitizer was also measured (black).
(B) Temporal change of fluorescence intensity of anthracene unit in
MSN-Anth. MSN-Anth was photoirradiated (665 nm) in the presence
of MSN-MB (0.1 mg/mL, 4.1 μM of MB units in MSN-MB, circle) or
MB-NH2 (4.1 μM, square). The control photoirradiation in the
absence of photosensitizer was also conducted (triangle).
that chemical modification of MB-NH2 by nanoparticles did
1
not interfere with the photochemical generation of O2.
To clarify the reason for this suppressed cytotoxic effect, we
1
then evaluated the generation and active area of O2 generated
Figure 2. Photoinduced cytotoxicity of MB-NH2 and MSN-MB
against A549 cells. After the cells were incubated for 24 h in the
presence or absence of MB-NH2 (41 μM) or MSN-MB (1.0 mg/mL,
41 μM of MB units in MSN-MB), the cells were irradiated (665 nm,
2.0 mW/cm2) for 0, 1, 3, and 6 h. The cell viability was calculated by
means of cell counting kit-8 (WST-8). Results are shown with the
mean SD (n = 3).
1
inside MSN-MB. First, we measured the quantum yield of O2
formation upon photoirradiation of MSN-MB and MB-NH2.
Detection of phosphorescence of 1O2 at 1270 nm revealed that
the quantum yield of MSN-MB was estimated to be 0.62, given
that the quantum yield obtained from MB-NH2 was similar to
that from methylene blue itself (ΦΔ = 0.52).23 Thus, MSN-MB
efficiently generated 1O2 under photoirradiation conditions.
1
1
the presence of MB-NH2 under conditions without photo-
irradiation. On the other hand, photoirradiation of the cells to
which MB-NH2 was administered resulted in a significant
decrease in cell viability, indicating generation of 1O2 by
photoirradiation of MB-NH2. We next conducted similar
cellular experiments using MSN-MB, in which the number of
MB molecules administered to the cells was equalized to that in
cells treated with MB-NH2 (Figure S1). In contrast to the
significant cytotoxic effects of MB-NH2, MSN-MB showed
almost no cytotoxic effect on the cells, even upon photo-
irradiation. In addition, we estimated the oxidative stress of the
cells. We measured the GSH/GSSG ratio in the cells treated
with MSN-MB upon photoirradiation. Although the photo-
irradiation caused slight decrease of the ratio (Figure S2), the
change was negligible, indicating that MSN-MB showed less
stress on the cells even upon photoirradiation. To elucidate
why MSN-MB caused no phototoxicity, we next assessed the
Second, we estimated the active area of O2. Because O2 is
known to become inactivated in cells within a distance of less
than 70 nm,24,25 we predicted that the active area of O2 was
1
limited to the pores of MSN. Active 1O2 was detected by means
of anthracene molecules tethered in the pores of MSN (MSN-
Anth; see Figure S3), the fluorescence of which was reduced by
the [4 + 2] cycloaddition of 1O2.26,27 Because anthracene units
tethered in the pores of MSN-Anth could not enter the pores of
MSN-MB, the 1O2 species generated inside the pores of MSN-
MB could not react with the anthracene units of MSN-Anth.
1
Therefore, we could track the presence or absence of O2
outside the pores of MSN-MB by monitoring the fluorescence
of MSN-Anth. We photoirradiated a mixture of MSN-Anth and
MSN-MB, and confirmed that the change in fluorescence
intensity of the anthracene units was negligible (Figure 3B). In
a control reaction, we compared the photoreaction of MB-NH2
or MSN-MB in the presence of monadelphous 2-amino-
anthracene, and confirmed that both photoreactions proceeded
immediately (Figure S4). The evidence that the drastic
fluorescence decay upon photoirradiation to a mixture
consisted of MB-NH2 and MSN-Anth led to the conclusion
1
generation of O2 by means of singlet oxygen sensor green
1
(SOSG), which is a fluorescent indicator of O2 (Figure 3A).
The cells, to which MSN-MB or MB-NH2 were administered,
were incubated with SOSG for 5 h, and then the cells were
photoirradiated for 1 h. We observed a dramatic increase of
fluorescence of SOSG from the cells, to which MSN-MB was
administered, while negligible increase of emission was
observed from the cells incubated without MSN-MB. Thus,
the 1O2 was produced in the cells upon photoirradiation, when
MSN-MB was administered to the cells, despite the fact that
cytotoxicity was undetectable. The experimental result that
fluorescence enhancement of SOSG in the presence of MB-
NH2 was lower than that in the presence of MSN-MB suggests
1
that O2 was generated mainly within the pores of MSN-MB
and was inactivated before leaking out of the pores. Thus, O2
1
species generated in the MSN-MB were confined to the pores,
resulting in reduced phototoxicity to living cells even under
photoirradiation conditions. We also confirmed that the 2-
aminoanthracene was oxidized by MSN-MB under the
conditions between pH 5.5 and 7.5; however, the reaction
efficiency was lowered probably due to the presence of
inorganic salt (Figure S5).
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Figure 4. (A) Formation of AQ from preAQ upon photoirradiation in the presence of MSN-MB. (B) Cell cycle phases of A549 cells. The cells were
incubated in the presence of designated reagents (preAQ: 10 μM, AQ: 10 μM, NaN3: 100 μM) and photosensitizer (MSN-MB: 1.0 mg/mL) for 24
h. After wash of the cells, the cells were irradiated (665 nm) for 0 or 3 h and followed by a further incubation for 24 h. The cell cycle phase was
analyzed by Nuclear-ID Green Cell Cycle Analysis KIT for flow cytometry. (C) Ratio of active caspase-9 to active caspase-3 in A549 cells, which
were treated by AQ, preAQ, MSN-MB, or MB-NH2 upon photoirradiation (665 nm, 0 or 3 h). After photoirradiation, the cells were further
incubated for 72 h and the ratio of active caspases was determined by immunofluorescence detection with APO Logix caspase detection kit.
Remote Photoregulation of the Cell Cycle. We then
shown in Figure 4B, the cell cycle phase was not detectably
changed compared with nontreated cells when the cells were
treated with MSN-MB without photoirradiation. It is striking
that photoirradiation of the cells treated with both MSN-MB
and preAQ resulted in a remarkable increase in the proportion
of cells in the G2/M phase. In separate experiments, we
attempted the remote photoregulation of cellular functions by
1
nontoxic O2 generation using the photoreactive MSN-MB
system. We chose the cell cycle and its arrest as the target for
evaluation of our system. It has been well-documented that
anthraquinone derivatives like doxorubicin and daunorubicin
induce G2/M cell cycle arrest by intercalation to double-
stranded DNA,28−32 and that they can be synthesized from
1
confirmed that addition of NaN3 as a O2 quencher led to the
suppression of this change in the cell cycle phase. These results
33,34
1
1
their corresponding anthracenes by O2 oxidation.
Based
strongly indicate that O2 generated in the pores of MSN-MB
on these contexts, we attempted to apply our system for in situ
generation of anthraquinone derivatives in the cells to initiate
cell cycle arrest (Figure 4). In living cells, preAQ, which has no
effect on the cells, would be converted to AQ by photoreaction
of MSN-MB without any side effects, and the resulting AQ
would lead to arrest of the cell cycle at G2/M phase.
in the cells reacted with preAQ to form AQ that arrested the
cell cycle. It should be noted that no cytotoxicity was observed
upon photoirradiation to the cells, which were treated with
MSN-MB and preAQ, respectively. Thus, photochemical
regulation of the cell cycle was accomplished without any
damage to the living cells.
Before the implementation of these cellular experiments, we
evaluated the chemical reaction of preAQ in the presence of
MB-NH2 upon photoirradiation by HPLC, and confirmed that
preAQ was efficiently converted to AQ (Figure S6). To
investigate the induction of G2/M cell cycle arrest by
photochemical production of AQ, A549 cells were pretreated
with both preAQ and MSN-MB for 24 h and then were
photoirradiated (665 nm) for 6 h after washing of the cells to
remove the unbound preAQ and MSN-MB. After further
incubation for 24 h, the cell cycle was examined using a flow
cytometer and a Nuclear-ID Green Cell Cycle Analysis kit. As
Identification of Photochemical AQ Production in the
Cells by Monitoring Caspase-3 and Caspase-9. To clarify
whether AQ was produced from preAQ in the cells, we
obtained cell extracts and tried to analyze the product. The
composition of the lysates was evaluated several times by
HPLC; however, identification of AQ production was
unsuccessful, probably because the amount of AQ was below
the measurable limit. Because we failed to detect the formation
of AQ by HPLC, we then attempted the identification of AQ
formation using biological methods. We evaluated the
activation of caspases, which are a family of cystein proteases
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that are markers of apoptosis. Recent reports demonstrated that
ROS, including O2, induce the activation of caspase-9 as well
arrested. Thus, the present photoirradiation system is applicable
to regulation of the cell cycle even in vivo.
1
as caspase-3, whereas the cell cycle arrest activates relatively
more caspase-3.35−38 Thus, identification of the species of
caspase activation could indirectly verify the generation of AQ
leading to cell cycle arrest in the living cells. We evaluated the
ratio of active caspase-9 to capase-3 by immunofluorescence
detection with an APO Active Detection kit. As shown in
Figure 4C, the cells incubated in the presence of MB-NH2
showed an increase of caspase-9 upon photoirradiation,
indicating that 1O2 was effectively generated and induced
apoptosis. In the case of the photoreaction system using MSN-
MB and preAQ, activation of caspase-3 was greater than that of
caspase-9. Control experiments using monadelphous AQ
showed that activation of caspase-9 was limited and that
caspase-3 was activated slightly, suggesting that photo-
irradiation of MSN-MB and preAQ generated AQ in the
cells, which resulted in arrest of the cell cycle at G2/M phase.
Cell Cycle Arrest in Vivo Experiments. Further attempts
were made to demonstrate the in vivo photochemical
regulation of cell cycle arrest using the present system. To
enhance the biocompatibility of the nanoparticles, polyethylene
glycol (PEG)-coated nanoparticles,24 which had MB units
incorporated in their pore (MSN-MB-PEG; see Figure S7)
were freshly prepared. For easy handling, we employed tumor
tissue as a target to evaluate cell behavior during photochemical
treatment. We transplanted colon 26 tumor cells subcuta-
neously into the lower thigh of nude mice. After the tumor
grew to an appropriate size, a solution of preAQ and MSN-MB-
PEG in saline (300 μL) was administered to the tumor tissue
by localized injection. Then, the tumor tissue was photo-
irradiated for 1 h. After irradiation, the mice were sacrificed and
the tumor cells were harvested immediately for cell cycle
analysis by flow cytometry. As shown in Figure 5, the cells
obtained from the irradiated mice were arrested in G2/M
phase, while those from the nonirradiated mice were not
CONCLUSION
■
Two major problems of conventional methods for the
regulation of cellular function by photoirradiation are the
generation of ROS during photoirradiation and their low
molecular selectivity in the chaotic environment of living cells.
First, to achieve effective resolution of the problem that ROS
strongly oxidize numerous biomolecules to damage living cells,
we utilized the pores of nanoparticles as a constrained reaction
1
field to produce confined O2. Upon photoirradiation, MSN
with photosensitizing MB units in their pore generated O2
1
exclusively within the pores, which could not leak out from the
pores due to its short lifetime. As a result, MSN-MB showed
dramatically reduced photocytotoxicity, because the photo-
chemical oxidation occurred predominantly in the pore.
Second, we attempted to use the pores of MSN for selection
of target molecules in the cells. The pore size successfully
selected small molecules from large biomolecules such as
nucleic acids or proteins, leading to selective photochemical
conversion of the small molecules.
This newly designed intracellular photoreaction system was
applied to photoregulation of the cell cycle. The cell cycle arrest
reagent, AQ, was synthesized in living cells by the oxidation of
the corresponding anthracene (preAQ) with singlet oxygen,
leading to arrest of the cell cycle only after photoirradiation
(Figure 4). Although the direct observation of intracellular AQ
production was unsuccessful because the amount of AQ
produced in the cells was small, it should be noted that we
observed AQ production indirectly by checking the apoptotic
pathway. We also confirmed that MSN-MB acted even in vivo
for control of cellular functions in tumor tissue.
The present system, which can produce photochemical
reactions in living cells without any side effects, offers new
perspectives for the field of photochemistry. The applications of
the system to photoregulation of single cell function and
organelle-specific photoregulation by means of appropriate
surface modification of MSN are in progress.
EXPERIMENTAL SECTION
■
General. All reagents were purchased from Nacalai Tesque
(Kyoto, Japan), NOF Corporation (Tokyo, Japan), Life
Technologies Japan (Tokyo, Japan), Wako pure Chemical
Industries (Osaka, Japan), Tokyo Chemical Industry (Tokyo,
Japan), or Sigma-Aldrich Japan (Tokyo, Japan), and were used
1
without further purification. H and 13C NMR spectra were
measured with a JEOL EX-400 or EX-300 spectrometer.
Electrospray ionization mass spectrometry (ESI-MS) were
conducted using a Thermo Fisher Scientific Exactive. Reversed
phase high-performance liquid chromatography (RP-HPLC)
was performed on a HITACHI D-2000 HPLC system using a
Cosmosil 5C8-MS column (5 μm, 4.6 × 250 mm) and the
elution peaks were detected using a UV−vis detector L-7455 at
300 nm. The solvent mixture of 0.1 M triethylamine acetate
(TEAA, pH 7.0) and acetonitrile was delivered as mobile phase
at a flow rate of 0.6 mL/min at 40 °C. UV−vis NIR adsorption
spectra were acquired on a JASCO V-530 spectrophotometer.
Fluorescence spectra were recorded on a Shimadzu RF-5300PC
spectrofluorophotometer. Transmission electron microscopy
(TEM) images were taken on a JEOL JEM-1400 operated at
120 kV electron beam accelerating voltage. Zeta potentials of
Figure 5. Cell cycle phases of colon 26 cells, which were collected
from tumor tissue transplanted in mice. After the solution of preAQ
(100 μM) and MSN-MB-PEG (10 mg/mL) in saline (300 μL) was
administerd to the tumor tissue by localized injection, photoirradiation
(665 nm) was conducted for 1 h. Then, the tumor cells were obtained
immediately to analyze cell cycle phase by Nuclear-ID Green Cell
Cycle Analysis kit for flow cytometry.
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1
dispersed nanoparticles in water were measured with a Malvern
Nano-ZS Zetasizer. Measurements were carried out in
triplicate, and the Smoluchowski approximation was used to
convert the electrophoretic mobility to zeta potential.
Fluorescence data in living cells were recorded using a Promega
(145 mg, 0.52 mmol, 48% over 2 steps): mp 90−92 °C; H
NMR (400 MHz, CDCl3) δ 9.28 (br, 1H), 8.45 (s, 1H), 8.32
(d, J = 10.2 Hz, 2H), 7.92 (d, J = 8.3 Hz, 3H), 7.46 (dd, J = 8.8,
2.0 Hz, 1H), 7.40 (m, 2H), 3.13 (s, 2H), 2.39 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 169.0, 134.3, 132.1, 131.8, 131.0,
129.1, 129.1, 128.1, 127.8, 125.9, 125.5, 125.4, 124.8, 120.3,
115.0, 63.6, 45.9; HR MS(ESI+) m/z: Calcd for C18H19N2O+:
39
GloMax Multi Detection System. MB-NH2 was synthesized
following a previously reported procedure. A confocal
microscopy (Zeiss LSM710) was used for in vitro studies.
Human lung carcinoma cell line, A549, was purchased from
American Type Culture Collection (Manassas, VA) and colon
26 was given from Prof. Michiyuki Matsuda and Dr. Yuji
Kamioka (Department of Medicine, Kyoto University). These
cells were maintained in Dulbecco’s Modified Eagle Medium
(Invitrogen Corp., Carlsbad, CA) containing 10% of fetal
bovine serum in a humidified incubator with 5% CO2, 95% air
at 37 °C. BALB/c nu/nu mice were purchased from SLC
(Shizuoka, Japan). The mice were housed at the Institute of
Laboratory Animals at Kyoto University Graduate School of
Medicine. All studies and procedures were approved by Animal
Research Committee of Kyoto University Graduate School of
Medicine. All animal experiments were performed according to
the Institutional Guidance of Kyoto University on Animal
Experimentation and under permission of the animal experi-
ment committee of Kyoto University.
+
279.1492 [M + H]+, found 279.1484 [M + H] .
Surface Modification of Mesoporous Silica Nanoparticles
(MSN). To a solution of MSN-CO2H (1.0 mg) in DMSO (100
μL) was added a mixture of MB-NH2 (595 μg, 1.1 μmol),
EDCI (170 μg, 1.1 μmol), and DMAP (13 μg, 0.11 μmol) in
DMSO (500 μL). The mixture was stirred at r.t. for 18 h. The
blue precipitate was produced by the addition of water (500
μL) and isolated by centrifuging at 13.2k rpm for 10 min.
Washing of this precipitate with water three times gave MSN-
MB as a dark blue powder. MSN-anth is also fabricated
following the above method from carboxylic-functionalized
MSN, an excess of 2-aminoanthracene and condensation
agents.
Synthesis of MSN-MB-PEG.40 To a solution of MSN-CO2H
(165 mg, 6.8 μmol of COOH units) in DMF (10 mL) were
added PEG-NH2 [MW 42 000] (10.3 mg, 0.24 μmol, 3.5%
equiv, ME-400EA from NOF corporation), N,N-dicyclohex-
ylcarbodiimide (DCC) (2.1 mg, 10 μmol), and DMAP (0.5 mg,
4.1 μmol). The reaction mixture was stirred at room
temperature for 18 h, and the resulting solutions was
centrifuged at 1500 rpm for 3 min in 15 mL tube to remove
the supernatant. Further washing with DMSO and water
afforded MSN with both PEG units and CO2H units (MSN-
CO2H-PEG) as a white powder. This highly hydrosoluble
nanoparticles was subsequently mixed with MB-NH2 (6 μmol)
in DMSO (5 mL) including EDCI (1.7 mg, 10.1 μmol) and
DMAP (0.2 mg, 1.6 μmol) at r.t. for 18 h. Water (3 mL) was
added to the resulting mixture and a blue precipitate was
produced. To isolate the blue precipitate, the mixture was
centrifuged at 3 krpm for 5 min and washed with water twice.
Drying under reduced pressure afforded MSN-MB-PEG as a
bright blue powder (54 mg). The formation of MSN-MB-PEG
was identified by measurement of UV spectra (see Figure S4).
Cell Culture. The cells were cultured at 37 °C in 5% CO2 in
75 cm2 flasks containing Dulbecco’s Modified Eagle Medium
(D-MEM) supplemented with 10% fetal bovine serum (FBS)
and 1% antibiotics.
Fluorescence Microscope Imaging. For fluorescence
imaging, the cells were plated in an imaging microplate (μ-Dish
35 mm) with a seeding density of 5 × 104 cells per well, and
preincubated for 24 h. The cells were further incubated for 3 h
with either MSN-MB (1.0 mg/mL) or MB-NH2 (41 μM) in
300 μL D-MEM. After washing with the medium (no Phenol
Red), the cells were observed with a confocal laser scanning
microscopy (Zeiss, LSM710) using appropriate excitation and
emission filters.
Cytotoxicity Assay (WST-8 Assay). The cytotoxicity
assays were performed in 96-well plates with a seeding density
of 1 × 104 cells per well. The cells were preincubated overnight
at 37 °C in 5% CO2 and then further incubated for 24 h with
the medium containing test substances. After incubation, 10 μL
of Cell Counting Kit-8 solution (Dojindo) was added to each
well and the plates were incubated for 3 h at 37 °C in 5% CO2.
Absorbance at 450 nm was recorded on a xMark microplate
spectrophotometer (BioRad).
Synthesis of N-(Anthraquinon-2-yl)-2-(dimethylamino)-
acetamide (AQ). A mixture of 2-aminoanthraquinone (761
mg, 3.41 mmol), 2-chloroacetyl chloride (1.0 mL), and pyridine
(0.8 mL) in DMF (1.0 mL) was stirred at r.t. for 18 h. The
resulting mixture was added into a small amount of crushed ice,
and the appearing precipitate was obtained by filtration. After
washing with water, the residue was extracted with CHCl3.
Concentration of the resulting solution gave N-(anthraquinon-
2-yl)-2-chloroacetamide as a brown solid. The chlorinated
anthraquinone derivative was added into the mixture of 50%
aqueous solution of dimethylamine (1.0 mL), pyridine (800
μL), and DMF (10 mL) without further purification and stirred
at 80 °C for 3 h. The resulting mixture was quenched with
water and then extracted with CH2Cl2. The obtained mixture
was concentrated under reduced pressure and column
chromatography (25−100% CHCl3/Hexane) of the residue
gave the desired product as a yellow powder (479 mg, 1.56
1
mmol, 46% over 2 steps): mp 141−143 °C; H NMR (300
MHz, CDCl3) δ 9.54 (br, 1H), 8.24 (dd, J = 8.8, 1.8 Hz, 1H),
8.16−8.13 (m, 3H), 8.02 (d, J = 1.8 Hz, 1H), 7.66 (t, J = 7.0
Hz, 2H), 3.08 (s, 2H), 2.36 (s, 6H); 13C NMR (75 MHz,
CDCl3) δ 182.6, 181.7, 169.3, 143.0, 134.4, 134.0, 133.6, 133.4,
133.3, 129.0, 128.9, 127.0, 126.9, 123.8, 116.3, 63.4, 45.9; HR
+
MS(ESI+) m/z: Calcd for C18H17N2O3 : 309.1234 [M + H]+,
+
found 309.1222 [M + H] .
Synthesis of N-(anthracen-2-yl)-2-(dimethylamino)-
acetamide (preAQ). A mixture of 2-aminoanthracene (210
mg, 1.09 mmol), 2-chloroacetyl chloride (413 mg, 3.7 mmol),
and triethylamine (120 μL) in dry THF (7.0 mL) was stirred at
r.t. for 18 h. The resulting mixture was concentrated under
reduced pressure and then extracted with ethyl acetate in the
presence of water. Concentration of the resulting solution gave
N-(anthracen-2-yl)-2-chloroacetamide as an orange−white
solid. To a solution of the chlorinated anthraquinone derivative
in dry THF (5 mL) was added dropwise 50% aqueous solution
of dimethylamine (1.0 mL) and stirred at 50 °C for 3 h. The
resulting mixture was concentrated under reduced pressure and
column chromatography (0−16% MeOH/CHCl3) of the
residue gave the desired product as a bright yellow powder
F
DOI: 10.1021/acs.bioconjchem.6b00061
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Quantification of Generated Singlet Oxygen in Cells.
Generated singlet oxygen was measured by using Singlet
Oxygen Sensor Green (SOSG) containing a fluorescein unit.
Cells were plated in 96-well plates with a seeding density of 1 ×
104 cells per well for 24 h and then incubated in the presence or
absence of MSN-MB (0.1 mg, 4.1 nmol MB units) in 100 μL
D-MEM including SOSG (25 μM) for 5 h. After washing with
D-MEM (no phenol red) three times, the cells were irradiated
by visible light (665 nm, 2.0 mW/cm2) for 0 or 1 h, and then
the fluorescence intensity of the cells was recorded on a
fluorescence plate reader (Promega GloMax Multi Detection
System) (Ex/Em: 490/510−570 nm).
Determination of GSH/GSSG Ratio. Levels of GSH and
GSSG were measured using a GSH/GSSG quantification kit
(Dojindo, Japan). Briefly, A549 cells were preincubated
overnight at 37 °C in 5% CO2 and further incubated for 24
h with the medium containing test substances. After washing
with DPBS three times, the cells were irradiated by visible light
(665 nm, 2.0 mW/cm2) for 30 min. The cells were harvested
and centrifuged for 10 min and the supernatant discarded. The
pellets were washed with 500 μL of cold DPBS and centrifuged
for 10 min at 4 °C and the supernatant discarded. After the
pellets were resuspended with 80 μL of 10 mM HCl, the cell
suspensions were frozen−thawed twice. Then, 20 μL of 5% 5-
sulfosalicylic acid (SSA) was added to the suspensions and
centrifuged for 10 min at 4 °C. The supernatants were diluted
with ultrapure water to reduce the SSA concentration to 0.5%
for assay. The samples were reacted with 5-mercapto-2-
nitrobenzoic acid and the absorbance at 405 nm was recorded
on a xMark microplate spectrophotometer.
in FITC channel of a flow cytometer with a 488 nm excitation
laser and a 530 nm detection filter. The obtained profiles were
analyzed in a Flowjo software (Tree Star) by Dean-Jett-Fox
fitting curve model.
Determination of Active Caspase 3 and 9. The ratio of
active caspase 9 to active caspase 3 was examined by
immunofluorescence detection with APO Logix caspase
detection kit (Cell Technology). Cell treatment by AQ (10
μM), MSN-MB (1.0 mg/mL, 41 μM MB), preAQ (10 μM), or
methylene blue (41 μM) were incubated for 24 h. After
washing of the cells, photoirradiation (665 nm, 2.0 mW/cm2)
was conducted for 3 h. All samples were further incubated for
72 h and the cell cycles were analyzed by detection kit.
Cell Cycle Photoregulation in Vivo. For the cell cycle
photoregulation in vivo, tumor bearing mice were subcuta-
neously injected MSN-MB-PEG (10 mg/mL) and preAQ (100
μM) in saline (300 μL). After 24 h, the mice were irradiated
(665 nm, 2.0 mW/cm2) for 1 h. Then, the mice were sacrificed
and the resected tumor cells were immediately dispersed by the
usual collagenase treatment. To analyze the cell cycle, the
obtained tumor cells were treated with Nuclear ID detection kit
and measured by using a flow cytometer as mentioned above.
Calculation of Inner and Outer Area of MSN Surface.
Inner and outer MSN surface area were calculated by eq 1 and
eq 2, respectively. Assuming that MSN (radius: 100 nm) is
composed of tubes (radius: 1.75 nm) hexagonally arranged in
28 layers (∼100/3.5) from the center, the inner and outer areas
were expressed in the following equations
2
⎛
⎞
⎟
⎠
⎛
⎞
⎟
1
2
2
⎜
⎜
Inner area = 1 × 2πr × 2 R − r 1 −
Active Area of Singlet Oxygen. Active singlet oxygen was
determined by using the fluorescence property of anthracene
tethered in the pore of MSN (MSN-Anth). MSN-Anth (0.2
mg) was stirred with either MSN-MB (0.1 mg, 4.1 nmol) or
MB-NH2 (4.1 nmol) in DMSO (1 mL) upon photoirradiation
(665 nm, 2.0 mW/cm2) for 0, 1, 2, 3, 5, 10, and 20 min. The
fluorescence intensities of the resulting samples were measured
on a Shimadzu RF-5300PC spectrofluorophotometer and
plotted (Ex/Em: 380/535 nm).
⎝
⎠
⎝
28
+ ∑ (6(n − 1)) × 2πr
n=2
2
⎛
⎞
⎟
⎠
⎛
⎞
⎟
1
2
2
⎜
⎜
× 2 R − r n −
⎝
⎠
⎝
(1)
(2)
Outer area = 4πR2
Generation of AQ from preAQ. After bubbling 100%
oxygen gas through a solution of preAQ (1 mM) and MB (41
μM) in water (1 mL) for 30 min, the solution was exposed to
visible light (665 nm, 2.0 mW/cm2) for 0, 1, 2, and 3 h. The
irradiated solutions (20 μL) were analyzed using reversed phase
HPLC. The detection wavelength was 300 nm and the
retention time of preAQ and AQ were 38.6 and 32.2 min,
respectively. After purification by HPLC, the formation of AQ
was identified by ESI-MS; HR MS(ESI−) m/z: Calcd for
where R = MSN radius (= 100 nm) and r = the pore radius (=
1.75 nm). Consequently, the inner area and outer area are 3.4 ×
106 nm2 and 1.3 × 105 nm2 per particle, respectively. Therefore,
the ratio of the inner area to the total area is approximately
96%.
ASSOCIATED CONTENT
■
S
* Supporting Information
−
C18H15N2O3 : 307.1088 [M-H]−, found 307.1091 [M-H]−.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.6b00061.
Cell Cycle Analyses on a Flow Cytometer. Cell cycle
analyses were performed following the instruction manual of
Nuclear-ID Green Cell Cycle Analysis Kit for Flow Cytometry
(Enzo Life Sciences). Cells were plated in 12-well plates with a
seeding density of 5 × 104 cells per well and then treated with
each reagent (10 μM) and/or MSN-MB (1.0 mg/mL) and/or
NaN3 (100 μM) in D-MEM (1 mL) for 24 h. After washing
with D-MEM (no phenol red) three times, the cells were
irradiated by visible light (665 nm) for 6 h and further
incubated for 24 h. The prepared adherent cells (∼1 × 105
cells) were trypsinized and centrifuged at 1000 rpm for 5 min
to remove the supernatant. The isolated cells were resuspended
in 0.5 mL of freshly prepared DNA Staining Solution,
incubated at 37 °C for 30 min in the dark, and then analyzed
Additional experimental results for characterization of
MSN-MB in the cells and photoreaction of preAQ, and
synthesis of MSN-MB-PEG (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-42-759-6229. FAX: +81-42-759-6493. E-mail:
tanabe.kazuhito@chem.aoyama.ac.jp.
Notes
The authors declare no competing financial interest.
G
DOI: 10.1021/acs.bioconjchem.6b00061
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
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ACKNOWLEDGMENTS
■
We sincerely thank Professor Teruyuki Kondo (Kyoto
University) for his valuable discussions and supports during
this study. We also thank Professor Tadashi Suzuki (Aoyama
Gakuin University) for valuable discussions about physical−
chemical properties of the compounds. This work is partly
supported by the Innovative Techno-Hub for Integrated
Medical Bioimaging Project of the Special Coordination
Funds for Promoting Science and Technology, and by Grant-
in-Aid for Scientific Research, from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan.
(20) Mondragon, L., Mas, N., Ferragud, V., de la Torre, C., Agostini,
A., Martinez-Manez, R., Sancenon, F., Amoros, P., Perez-Paya, E., and
Orzaez, M. (2014) Enzyme-responsive intracelular-controlled release
using silica mesoporous nanoparticles capped with ε−poly-L-lysine.
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