Development of a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles

Article abstract of DOI:10.1039/c4tb00536h

To effectively and safely use molecules, it is important to be able to control the timing and site of molecule activation. We developed a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles that can be prepared under mild conditions. The function of various molecules, such as rhodamine B, Nile blue A, propidium iodide (PI), and rhodamine 110, bis-(N-CBZ-l-arginine amide), dihydrochloride (BZAR), was restricted by wrapping in the network structure of the nanoparticle gel. The encapsulated molecule was released from the gel by the light stimulus and its function was restored. Hence, this technique is applicable to the functional control of various molecules. The PI-encapsulated nanoparticles were internalized by the cells after being conjugated with the cell membrane permeability peptide, octaarginine, and were localized to the cytoplasm. Short-term irradiation (20 s) induced PI release from the nanoparticles and the rapid movement (less than 2 min) of the released PI to the nucleus. These nanoparticles are thus useful tools for the spatiotemporal control of various molecular functions because they permit the quick and transient release of encapsulated molecules after short-term irradiation and can be prepared under mild conditions. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4tb00536h

Journal of
Materials Chemistry B
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PAPER
Development of a spatiotemporal method to
control molecular function by using silica-based
photodegradable nanoparticles
Cite this: J. Mater. Chem. B, 2014, 2,
4153
Fumi Ishizuka,a Xiangsheng Liu,b Shuhei Murayama,†a Tomofumi Santaa
a
*
and Masaru Kato
To effectively and safely use molecules, it is important to be able to control the timing and site of molecule
activation. We developed a spatiotemporal method to control molecular function by using silica-based
photodegradable nanoparticles that can be prepared under mild conditions. The function of various
molecules, such as rhodamine B, Nile blue A, propidium iodide (PI), and rhodamine 110, bis-(N-CBZ-L-
arginine amide), dihydrochloride (BZAR), was restricted by wrapping in the network structure of the
nanoparticle gel. The encapsulated molecule was released from the gel by the light stimulus and its
function was restored. Hence, this technique is applicable to the functional control of various molecules.
The PI-encapsulated nanoparticles were internalized by the cells after being conjugated with the cell
membrane permeability peptide, octaarginine, and were localized to the cytoplasm. Short-term
irradiation (20 s) induced PI release from the nanoparticles and the rapid movement (less than 2 min) of
the released PI to the nucleus. These nanoparticles are thus useful tools for the spatiotemporal control
of various molecular functions because they permit the quick and transient release of encapsulated
molecules after short-term irradiation and can be prepared under mild conditions.
Received 4th April 2014
Accepted 23rd April 2014
DOI: 10.1039/c4tb00536h
www.rsc.org/MaterialsB
molecule is physically wrapped by the network structure of the
gel, no chemical bonding is required for encapsulation using
Introduction
this technique. In addition, the mesh size of the gel can be
modied by changing the size of the monomer, and the size
range of the encapsulated molecule is broad (from small
molecules like uorescein (MW 389 Da) to large molecules like
ferritin (MW 450 kDa)).8,9 We thought that this technique was
applicable for the functional control of a variety of molecules
and named it the “PARCEL” method (Scheme 1). Furthermore
we found that the gel comprised 100 nm spherical
Each molecule has its own function(s) and modern society
makes use of millions or perhaps billions of functional mole-
cules. It is therefore essential to control the function of these
molecules spatiotemporally for their use in daily life, because
their activation at the wrong place or time can sometimes lead
to harmful effects.1,2 To control molecular function, external
stimuli (e.g. light, heat, or a magnetic eld) and stimulus-
sensitive elements or groups have been used.3,4 Light has long
been considered a suitable stimulus because of its excellent
usability and selectivity, and many elements and molecules
that are activated by light have been developed and used around
the world.5
We previously developed a universal method to functionally
control molecules by using a polyethylene glycol (PEG)-based
photodegradable gel.6,7 The molecular function can be
restricted when the molecule is encapsulated within the gel and
the activity is restored by releasing the molecule from the gel
when collapse of the gel is induced by irradiation. Because the
aGraduate School of Pharmaceutical Sciences and GPLLI Program, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bCenter for Medical Systems Innovation Summer Internship Program, The University of
Tokyo, Japan
† Present address: Molecular Imaging Center, National Institute of Radiological
Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Japan.
Scheme 1 Synthesis pathway of the photolinker and an image of
functional molecule encapsulation and release.
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nanoparticles when it was prepared by using a vortex for mix- nitrophenoxy]butyric acid was added at 75 ꢀC. Aer 48 h at 80 ꢀC
ing.10–12 Because photodegradable nanoparticles can serve as reux, the solvent was evaporated and the compound was
vehicles to transport a molecule to a target place and release the puried by column chromatography using silica gel and 10%
cargo molecule at that place in response to a photo stimulus, potassium carbonate.17 A mixture of ethyl acetate–hexane ¼
the molecule-encapsulated nanoparticles were considered 3 : 1(v/v) was used as an eluent, and a yellow product was
useful tools for the spatiotemporal control of molecular func- obtained.
tion. The quick and transient release of encapsulated molecules
1H NMR (500 M Hz, CDCl3): d ¼ 7.56 (s, aromatic-H), d ¼ 6.98
was also accomplished by irradiation, because collapse of the (s, aromatic-H), d ¼ 6.34 (q, aromatic-CH(CH3)), d ¼ 4.10 (t,
gel could be induced with a short burst of irradiation (as little as aromatic-OCH2), d ¼ 3.93 (s, aromatic-OCH3), d ¼ 3.81 (q,
10 seconds). Hence, the nanoparticles allow us to induce drastic Si(OCH2CH3)3), d ¼ 3.26 (m, aromatic-CHO(C]O)NHCH2), d ¼
changes in the concentration or the amount of active molecules 3.16 (m, aromatic-OCH2CH2CH2CONHCH2), d ¼ 2.35 (m,
at the time of irradiation. We used nanoparticles to locally aromatic-OCH2CH2CH2CONH),
d
¼
2.17 (t, aromatic-
activate molecules within a cell and tried to clarify the effect of OCH2CH2CH2CONH), d ¼ 1.62 (dt, CH2CH2Si(OCH2CH3)3), d ¼
releasing that molecule within the cell; we also tried to control 1.57 (d, aromatic-CHCH3), d ¼ 1.25 (t, Si(OCH2CH3)3), d ¼ 0.62
cellular functions using the released molecule.10–12
(t, CH2Si(OCH2CH3)3).
However, our recent studies indicate that some small mole-
cules, such as Nile blue A and propidium iodide (PI), are not
controlled correctly by the PARCEL method. We propose two
hypotheses to explain the lack of control of some small mole-
cules with the PARCEL method: (1) the mesh size of the gel
prepared from PEG was too large to encapsulate the small
molecule, or (2) the radical polymerization that was used to
prepare the gel damaged the small molecule during prepara-
tion. To test these hypotheses, we examined silica gel prepared
from the polymerization reaction of alkoxysilane. Because silica
has excellent biocompatibility, like PEG, and the mesh size of
silica gel was small, silica-based photodegradable gel could be
broadly applicable in this context. Moreover, our previous
research suggested that silica gel offers the advantages of
improved stability of the encapsulated molecule within the
silica gel and that it can be prepared under mild conditions.13–16
Therefore, in the present study we developed silica-based
photodegradable nanoparticles and applied them to the
spatiotemporal control of molecular function within the cell.
Preparation of seed and nanoparticles
The seed and nanoparticles were prepared based on the method
reported previously.18,19 Typically, 9.1 mg arginine was solved in
6.9 mL water and then 0.45 mL cyclohexane and 0.55 mL TEOS
were added to the solution in this order. The solution was
stirred at 60 ꢀC for 20 h and seeds with uniform size were
prepared.
The solution of 100 mL seed, 100 mL arginine (10 mg mLꢁ1) in
watꢁer1, and encapsulated molecules (40 mL rhodamine B (10 mg
mL ) or 100 mL Nile blue A (1 mg mLꢁ1) or 300 mL PI (50 mg
mLꢁ1)) were added to 3 mL water. Then, 15 mg photolinker in
ethanol (100 mL) and 35.2 mL TEOS were added. The mixture
ꢀ
solution was stirred with a speed of 300 rpm at 60 C for 24 h
and the diameter of the nanoparticles increased up to about 50
nm. The prepared nanoparticles were ltered through a viva-
spin centrifugal lter (molecular weight cut off of 30 k, Sarto-
¨
rius, Gottingen, Germany) to remove excess molecules which
were not encapsulated in nanoparticles.
Experimental section
Materials
Surface modication of photodegradable silica nanoparticles
0.25 mL of the nanoparticle solution and 0.25 mL of R8 peptide
(4 mM in water) were mixed and vortexed for 20 min at room
temperature.
Arginine, Nile blue A, rhodamine B, 3-(triethoxysilyl)propyl
isocyanate (IPS), dibutyltin(IV) dilaurate (DBT), hexane, potas-
sium carbonate, DNA from salmon sperm and ethyl acetate were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
4-[4-(1-Hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid and
octaarginine (R8) peptide were obtained from Sigma-Aldrich (St.
Louis, MO). Tetraethyl orthosilicate (TEOS) was from Kojundo
Chemical Laboratory (Saitama, Japan). Cyclohexane was from
Tokyo Chemical Industry (Tokyo, Japan). Silica gel (0.063–0.210
mm) was from Kanto Chemical co., inc, (Tokyo, Japan). SYTO16,
and rhodamine 110, bis-(N-CBZ-L-arginine amide), dihydro-
chloride (BZAR) were from Invitrogen (Carlsbad, CA). Propidium
iodide (PI) was from Dojindo (Kumamoto, Japan). Water was
puried with a Milli-Q apparatus (Millipore, Bedford, MA).
Transmission electron microscopy (TEM) observation
TEM images were obtained with an H-7000 electron microscope
(Hitachi, Tokyo, Japan) operating at 75 kV. Copper grids (400
mesh) were coated rst with a thin lm of collodion and then
with carbon. The nanoparticle dispersion (5 mL) was placed on
the coated copper grids.
Diameter and zeta potential measurements by dynamic light
scattering (DLS)
Delsa Nano (Beckman Coulter. Inc. Brea CA, USA) was used for
the measurement of diameters and zeta potentials of the
prepared nanoparticles. The nanoparticle solution was diluted
Synthesis of photolinker (Scheme 1)
IPS (1.34 mmol) and DBT (0.033 mmol) were dissolved in by water for diameter measurement or 10 mM NaCl solution for
anhydrous ethyl acetate, then 4-[4-(1-hydroxyethyl)-2-methoxy-5- zeta potential measurements.
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Irradiation conditions
Results and discussion
Preparation of photodegradable nanoparticles containing
UV curing equipment, Aicure UV20 (Panasonic, Osaka, Japan),
was used as a light source for UV irradiation. The wavelength of
rhodamine B
the irradiating light was 365 nm at an intensity of 20 mW cmꢁ2
.
First, we prepared silica-based photodegradable nanoparticles
containing rhodamine B. The seed particles (27 nm in diameter)
were prepared by the hydrolysis and condensation reactions of
TEOS. Then the seeds grew by the addition of rhodamine B,
photolinker, and TEOS to the seed solution. To examine the
inuence of the nanoparticle composition on the release of the
encapsulated molecule, we prepared two different nano-
particles: one containing the photolinker, and the other without
the photolinker. The size and shape of nanoparticles were
analyzed by means of DLS and TEM (Fig. 1). The shape, size
distribution, and average size of the nanoparticles with or
without the photolinker were similar. Both types of nano-
particles were spherical in shape and their size distributions
were narrow (both types of particles were about 50 nm). These
results indicate that the addition of the photolinker did not
affect the formation of the nanoparticles.
Then, we compared the amount of rhodamine B released
from these rhodamine B-encapsulated nanoparticles before and
aer UV irradiation (Fig. 2). The supernatant of rhodamine B for
both types of nanoparticles showed very low uorescence
intensity before UV irradiation. However, aer irradiation, the
uorescence intensity was increased (about 7 times) only for the
rhodamine B encapsulated in the nanoparticles with the pho-
tolinker. Then, the excitation and emission spectra of rhoda-
mine B, rhodamine B-encapsulated nanoparticles, and released
rhodamine B were compared. Although small shis of the
maximal excitation and emission wavelengths were observed
The irradiation times of the nanoparticles in a 0.6 mL micro-
tube and those in cells were 1 min and 20 s, respectively.
Measurement of released molecules
Aer the irradiation, the nanoparticle solution was centrifuged
at 120 000 rpm for 30 min using ultracentrifuge equipment
(Optim TLX, Beckman Coulter, Inc. Brea, CA). The supernatants
of the solution (100 mL) were moved to wells of a microplate. 100
mL of 10 mM phosphate buffer (pH 7.4) for rhodamine B and
Nile blue A assays or 100 mL of 0.2 mg mLꢁ1 DNA solution for
the PI assay was added to each well before uorescence
measurement. The uorescence (ex 570 nm, em 590 nm for
rhodamine B: ex 635 nm, em 674 nm for Nile blue A: ex 530 nm,
em 620 nm for PI: ex 480 nm, em 520 nm for BZAR) was
measured with a multiplate reader (SH-9000, Corona Electric
Co., Ibaraki, Japan).
Cell culture and the nanoparticle internalization to cells
The adenocarcinomic human alveolar basal epithelial (A549)
cells were cultured in a humidied atmosphere of 95% air and
5% CO2 at 37 ꢀC in Dulbecco's Modied Eagle's Medium
(DMEM, Sigma-Aldrich) supplemented with 1% (v/v) penicillin–
streptomycin solution (ꢂ100) (Wako) and 10% (v/v) fetal bovine
serum (FBS, Invitrogen). For each experiment, cells at 80–90%
conuence were harvested by trypsin/EDTA (Wako) digestion,
washed and re-suspended in fresh growth medium with FBS at a
cell concentration of 106 viable cells per culture dish (75 cm2).
All cells were cultured in a 35 mm glass bottom dish.
For the internalization of the nanoparticles to cells, the
cultured dish was washed by DMEM without FBS. Then, 200 mL
of the nanoparticle solution was dropped to the dish and the
dish was allowed to stand for 1 hour at 37 ꢀC for internalization.
Aer that, the sample was washed 2 times by PBS. Then the
SYTO16 dye was added to the medium at a nal concentration
of 500 nM to stain the nucleus and incubated for 20 min at 37
ꢀC. The cells were washed 3 times with PBS and were supplied
fresh DMEM before irradiation.
Microscopy observation
The confocal uorescence microscopy images of the cells were
obtained with an FV10i microscope (Olympus, Tokyo, Japan).
The laser power of the microscopy was set at 40%.
Fluorescence intensity analysis in the nucleus
Fluorescence intensity within the nucleus was evaluated by
means of confocal laser scanning microscopy. The uorescence
intensity in the images was analyzed with ImageJ soware (ver.
1.45s; http://rsbweb.nih.gov/ij/index.html). The mean gray
value of each nucleus was calculated from three different areas
in a nucleus. The average values were obtained from the mean
gray values of 5 different cells.
Fig. 1 (a) DLS and (b) TEM analysis of seed and nanoparticles with and
without the photolinker, and (c) excitation and emission spectra of free
rhodamine B, rhodamine B-encapsulated nanoparticles, and released
rhodamine B.
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irradiation (Fig. 2). The irradiation increased the uorescence
signals of the supernatants by 7- and 3-fold for Nile blue A and PI,
respectively. The amounts of released Nile blue B and PI were
estimated to be about 13 and 5%, respectively, these molecules
remained in the nanoparticles aer irradiation. It was supposed
that this low release efficiency of PI originated from many posi-
tive charges of PI than those of rhodamine B and Nile blue A.
The effect of the UV intensity on the release amount was
examined. Four different UV intensities (30, 60, 120, and 250
mW) were used to study. Fig. 2b shows the relationship between
the light intensity and the released amount observed aer the
irradiation. Predictably, the largest quantity was obtained when
the strongest intensity of UV light was used (250 mW), and the
quantity decreased with decreasing light intensity. A change of
the nanoparticles that was derived from UV irradiation was
observed by TEM observation (Fig. 2c). Although the nano-
particle before the irradiation was spherical in shape and the
surface looked like smooth, the surface of the nanoparticle aer
the irradiation became rough. It was supposed that this rough
surface was derived from many cracks formed by cleavage
reaction of the nitrobenzyl group. These data suggest that silica
nanoparticles could be used for the controlled release of various
molecules, because small molecules whose release was difficult
to control using PEG-based nanoparticles could be released
from silica nanoparticles by use of irradiation.6 This was derived
from the mild preparation conditions of the silica nanoparticles
and the small mesh of the nanoparticles.
Fig. 2 (a) Release of the encapsulated molecule from the nano-
particles with or without irradiation, (b) relationship between irradia-
tion power and released BZAR from the nanoparticles, and (c) TEM
images of nanoparticles before and after irradiation.
Surface modication of the nanoparticles
for the nanoparticle, almost no shi of them was observed
between the original rhodamine B and released rhodamine B
(Fig. 1). These results demonstrate that rhodamine B encapsu-
lated in the photodegradable nanoparticles could be released
upon UV irradiation. Because the encapsulated molecules were
immobilized by the mesh structure that was prepared by the
polymerization reaction of the silica, this technique could be
applicable to various kinds of molecules for their encapsulation
and subsequent release by irradiation. Because the amount of
released rhodamine B was estimated to be about 15%, most of
Nanoparticles containing a functional molecule can be used as
vehicles to transport that molecule, because nanoparticles can
be delivered to various locations. Aer the nanoparticles reach a
target site, the encapsulated molecule can be released and
activated by light stimuli. Silica-based nanoparticles have been
used to carry functional molecules into cells and the molecules
within the cells were then activated by irradiation because silica
was thought to be a low immune response material. However,
silica nanoparticles are not internalized into cells without rst
being modied with a membrane permeable peptide, such as
R8 or HIV-1 Tat, to increase their uptake efficiency.20–22 We
analyzed the shape, size and zeta potential of the modied
nanoparticles by using TEM and DLS (Fig. 3a and b). The shape
of the nanoparticles was not changed by modications (Fig. 1
the rhodamine
irradiation.
B remained in the nanoparticles aer
Generality of the release technique
To examine the generality of this method, we also examined the and 3a), but the diameter range increased and the average
controlled release of Nile blue A and PI, the release of which are diameter of the nanoparticles became about 60 nm. The values
difficult to control using the PEG-based photodegradable gel.8 of the polydispersity index (PDI) of these nanoparticles were
The color of these compounds disappeared when they were increased from 0.083 to 0.231. The modication with the R8
encapsulated in PEG-based photodegradable nanoparticles, peptide produced a large difference in the zeta potential; the
because of the reaction with ammonium peroxodisulfate. The value was ꢁ29 ꢃ 6 mV before this modication and +15 ꢃ 1 mV
released amount of Nile blue A was measured from the uo- aer the modication. This charge difference of the nano-
rescence intensity of the supernatant. The released amount of particles increased their affinity for the cell surface, which is
PI from the nanoparticles was measured by the uorescence negatively charged. These results indicate that the surface of the
intensity of the complex of DNA and PI in the supernatant, nanoparticles was modied by the R8 peptide.
because the uorescence intensity of PI is changed by its
We then compared the internalization efficiencies of the
intercalation with DNA. The release of these compounds from nanoparticles with and without R8 modication. Both types of
the nanoparticles into the supernatant was observed aer nanoparticles containing PI were prepared and added to the
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Fig. 4 Fluorescence images of A549 cells treated with nanoparticles
containing PI. Upper and lower lanes show irradiated and nonirradi-
ated cells.
with silica gel via electrostatic interactions even aer the
collapse of the nanoparticles. No change was observed with the
nonirradiated cells; the red uorescence remained in the cyto-
plasm of the cells (Fig. 4 lower lane). In general, PI localizes to
the nucleus and intercalates with DNA when it is internalized
into a cell,23 but the PI within the nanoparticles could not reach
the nucleus when it was wrapped in 60 nm-sized nanoparticles,
because the PI-encapsulated nanoparticles could not penetrate
the 10 nm-sized nuclear pore.24 When the PI was released from
the nanoparticles by irradiation, the PI molecules could pass
through the nuclear pore and intercalate with DNA, causing the
red uorescence seen in the nucleus. In addition, the light
irradiation did not cause serious damage to the cells, because
our cell survival assessment showed no substantial changes.
Therefore, these data demonstrate that photodegradable silica
nanoparticles can be used to control the release of encapsulated
molecules within a cell.
Fig. 3 (a) TEM and (b) DLS analysis of nanoparticles modified with the
R8 peptide, (c) fluorescence images of A549 cells treated with PI-
encapsulated nanoparticles modified with the R8 peptide or without
modification, and (d) the effect of nanoparticles and UV irradiation on
cell survival.
culture medium. The internalization efficiency of the unmodi-
ed nanoparticles was low, because less uorescence signal was
detected from the cells that were treated with the unmodied
nanoparticles (Fig. 3c). On the other hand, most of the cells that
were treated with the R8-modied nanoparticles showed uo-
rescence. This high internalization efficiency of R8-modied
nanoparticles is consistent with previous reports.20–22 The effect
of UV irradiation and nanoparticles on the cell activity was
examined using the MTT assay. No deleterious effects by the
irradiation and nanoparticles were observed (Fig. 3d).
Finally, we examined the time course of the uorescence
changes in the nucleus. We found that the red uorescence
signal in the nucleus started to increase within 2 min aer the
irradiation treatment and became stable at around 8 min
(Fig. 5). No further changes in uorescence intensity were
Release of the encapsulated molecule within cells by
irradiation
Then we tried to release the encapsulated molecule from the
nanoparticles by using light stimuli within the cells. To observe
the effect of the released molecule within the cell, we used
nanoparticles containing PI. Red uorescence was observed in
the cytoplasm of the cells before the irradiation when the cells
were treated with PI-encapsulated nanoparticles (Fig. 4). A
strong signal could not be detected in the nucleus where PI
generally localizes. This nding indicates that only the PI that
was encapsulated in the nanoparticles was internalized into the
cells. Aer irradiation, the red uorescence signal was detected
not only in the cytoplasm but also in the nucleus of the cells
(Fig. 4 upper lane). This signal was derived from PI that was
released from the nanoparticles and moved into the nucleus.
The uorescence signal that was still detectable in the cyto-
plasm was likely derived from PI that coexisted with degraded
Fig. 5 Time-dependent fluorescence changes in the nucleus of A549
nanoparticles, because PI is a cationic compound that interacts cells after with or without irradiation.
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observed in the nucleus for more than one hour. Because our
previous study showed that molecules released from nano-
particles within the cell distribute throughout the cell and reach
an equilibrium state within 10 min,9 the 8 min timeframe for
the distribution of the released PI by diffusion and subsequent
localization to the nucleus seemed reasonable. The release of
the PI was quick and transient, because the uorescence
intensity in the nucleus stabilized quickly. To examine the effect
of the releasing molecule within a cell, a rapid and transient
release is necessary because the effect disappears within the
approximately 10 min timeframe it takes to reach an equilib-
rium state. We believe that these nanoparticles represent a very
promising tool to clarify the effect of released molecules within
cells and to control cellular functions using released molecules.
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Conclusions
We developed a method to control molecular function by using
photodegradable silica-based nanoparticles. The nanoparticles
were effective tools to functionally control small molecules,
because the mesh size of the nanoparticle gel was small and no
radical polymerization was needed to prepare the nano-
particles. Surface modication of the nanoparticles with R8
permitted the nanoparticles to be internalized strongly into the
cells and deliver the encapsulated molecule to the cytoplasm.
The nanoparticles released the encapsulated molecule quickly
and transiently on exposure to a short burst of irradiation.
Given that many researchers are interested in the local cellular
functions of molecules, these nanoparticles should be broadly
applicable to such research. Drug delivery systems and photo-
activated sensors are other promising areas for the application
of nanoparticles, because the timing of release can be
controlled precisely with light stimuli.
Acknowledgements
We thank Dr S. Fukuda (U. Tokyo), Mr T. Tsuboya (U. Tokyo)
and Dr N. Kaji (Nagoya Univ.) for technical assistance in TEM
measurement, in cell study and in donating the R8 peptide,
respectively. This work was supported by grants (Kakenhi) from
the Ministry of Education, Culture, Sports, Science, and Tech-
nology (MEXT) of Japan, JSPS Core-to-Core Program, A.
Advanced Research Networks, and the Naito Foundation.
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4158 | J. Mater. Chem. B, 2014, 2, 4153–4158
This journal is © The Royal Society of Chemistry 2014