Spatiotemporal activation of molecules within cells using silica nanoparticles responsive to blue-green light

Article abstract of DOI:10.1039/c5tb01165e

The spatiotemporal control of molecular function is important but there are currently few techniques for noninvasively controlling various types of molecules in live cells. Herein we developed nanoparticles with a boron dipyrromethene structure, which are

Full text of DOI:10.1039/c5tb01165e

Journal of
Materials Chemistry B
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Spatiotemporal activation of molecules within
cells using silica nanoparticles responsive to
blue-green light
Cite this: J. Mater. Chem. B, 2015,
3, 7427
Takaki Amamoto,a Tomoya Hirata,a Hironori Takahashi,b Mako Kamiya,b
Yasuteru Urano,abc Tomofumi Santaa and Masaru Kato*a
The spatiotemporal control of molecular function is important but there are currently few techniques for
noninvasively controlling various types of molecules in live cells. Herein we developed nanoparticles
with a boron dipyrromethene structure, which are responsive to blue-green visible light. Fluorophores
(fluorescein, rhodamine B, and Nile blue A) encapsulated within the nanoparticles were released by
irradiation for 3 min with visible light. Nanoparticles were internalised by HeLa cells without the aid of a
cell-penetrating peptide, serving as vehicles for the delivery of cargo molecules to the cytoplasm. The
release and activation of encapsulated molecules by visible light irradiation demonstrate a novel method
for the spatiotemporal control of molecular function that can be used to activate molecules inside the
skin that cannot be reached by UV light, which has limited tissue penetration.
Received 17th June 2015,
Accepted 11th August 2015
DOI: 10.1039/c5tb01165e
www.rsc.org/MaterialsB
that is, the interaction between encapsulated and outside
molecules was hindered by the mesh structure of the gel. The
Introduction
Analytical techniques such as fluorescence lifetime imaging functional capacity was recovered with the release of the
and stochastic optical reconstruction microscopy1 have been molecule upon collapse of the mesh structure induced by UV
applied to the investigation of the kinetics and localization of irradiation. In this method, there was no requirement of
intracellular molecules.2 Because the location and timing of specific functional groups that can form a chemical bond with
intracellular molecule activation are precisely controlled in the gel framework for encapsulation, since the molecule was
cells, much attention has been focused on the methods for physically trapped by the mesh;10 moreover, there was no size
controlling molecular function within cells by using a non-invasive limit for encapsulation since the gel mesh size could be altered
external signal.3 To date there are few general techniques that can by adjusting the size of the monomer.11 Using this method, we
be used for this purpose.4–6
were able to control the function of proteins, small molecules,
Molecular activation using a non- or minimally invasive and short interfering RNA within cells, demonstrating its wide
external physical stimulus such as electric current or light, applicability.12,13 The collapse of the mesh structure occurred
temperature, and magnetic fields is of great interest in basic via the photoelimination of the nitrobenzyl group induced by
and applied research (for drug delivery, imaging, organic irradiation with UV light (ca. 350 nm). However, the applicable
synthesis, and so on).7,8 Light is among the most versatile types area of this technique was limited due to the low tissue
of stimuli since it is relatively safe and the timing, location, and penetration of UV light.14 Furthermore, the possibility of DNA
duration of irradiation can be precisely controlled. We previously damage is an important concern associated with the release of
developed an ultraviolet (UV) light-cleavable monomer with a molecules within cells via UV irradiation.15 Therefore, a technique
nitrobenzyl group to generate light-responsive nanoparticles in that makes use of longer-wavelength light is desirable.
the form of a photodegradable gel that collapses upon irradia-
Upconversion has been used to increase the wavelength of a
tion with UV light.9 These particles, which were used to control light source. The photoelimination reaction proceeds by the
molecular function in cells, were restricted by encapsulation; sequential absorption of two or more photons that are derived
from longer-wavelength light,16 and upconversion occurs with-
out changes to the photocleavable group when the wavelength
of light increases by two-fold or greater. However, high-powered
light is required for photoelimination, which is associated
with safety issues. The photoelimination reaction of 4-aryloxy
boron dipyrromethene (BODIPY) derivatives upon irradiation
a Graduate School of Pharmaceutical Sciences, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: masaru-kato@umin.ac.jp
b Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
c AMED CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan
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with blue-green visible light (B500 nm) has recently been Preparation of a BODIPY-based photocleavable monomer
reported.17,18
The preparation of photocleavable monomer is outlined in
Scheme 1. Benzaldehyde (256 mg) and 2,4-dimethylpyrrole
The reaction proceeds via photoinduced electron transfer,
which induces solvolysis of the B–O bond of the positively
(427 mg) were dissolved in 300 ml of DCM. Three drops of
charged aryl group.17 Since the reaction proceeds at ambient
trifluoroacetic acid were added and the mixture was stirred for
irradiation power, photoelimination of the BODIPY structure is
20 h at room temperature. 2,3-Dichloro-5,6-dicyanobenzoquinone
expected to be relatively safe. We hypothesized that visible
(720 mg) was added to the reaction solution, followed by stirring for
light-responsive gel could be prepared from a monomer with
10 min at room temperature. The reaction was washed twice with
a BODIPY structure instead of a nitrobenzyl group, and in this
water and brine and dried over Na2SO4. The crude product was
study, we developed silica nanoparticles that can be used to
purified by column chromatography over silica gel and the DCM
deliver molecules to cells and can be spatiotemporally controlled
fraction was evaporated under reduced pressure. The obtained
by visible light irradiation. This is the first report describing
solid was dissolved in 120 ml of toluene, followed by the dropwise
the control of various types of molecules in live cells by using
addition of 2.4 ml of N,N-diisopropylethylamine and 3.0 ml of
boron trifluoride. The solution was stirred for 2 h at room
nanoparticles stimulated by blue-green light.
temperature under a nitrogen atmosphere, washed twice with water
and brine, and purified by column chromatography over a silica gel
(n-hexane : ethyl acetate = 3 : 1). The yield for product 1 was 41%
Experimental
Chemicals
(324 mg).19
The reaction product (324 mg of 1) along with 66.7 mg of
aluminium chloride was dissolved in 30 ml of DCM and refluxed at
55 1C for 5 min under an argon atmosphere. 4-Hydroxybenzaldehyde
(266 mg) in 20 ml of DCM was added to the reaction using a syringe,
and the solution was refluxed for 5 min. The reactant was
purified using an alumina filtrate column followed by column
chromatography over a silica gel (n-hexane: ethyl acetate = 3 : 1),
then dried using an evaporator. The yield for product 2 was 42%
(220 mg); this was dissolved in 40 ml of methanol, to which
sodium borohydride (63 mg in 10 ml of methanol) was added
using a syringe, followed by stirring for 5 min at room temperature
under a nitrogen atmosphere. After adding 1 ml of acetone, the
crude product was purified by column chromatography over silica
gel (n-hexane : ethyl acetate = 1 : 1), yielding 140 mg of product 3
(63%). This was dissolved in 30 ml of ethyl acetate with stirring
under a nitrogen atmosphere. 3-Isocyanatopropyltriethoxysilane
Benzaldehyde, 2,3-dichloro-5,6-dicyano-p-benzoquinone, N,N-
diisopropylethylamine, aluminium chloride, dichloromethane
(DCM), sodium sulphate, toluene, n-hexane, ethyl acetate, 4-hydroxy-
benzaldehyde, sodium cyanoborohydride, acetone, L(+)-arginine
sodium chloride, rhodamine B, 40,6-diamidino-2-phenylindole
(DAPI), and methanol were purchased from Wako Pure Chemical
Industries, Ltd (Osaka, Japan). 2,4-Dimethylpyrrole, boron trifluoride,
3-isocyanatopropyltriethoxysilane, dibutyltin dilaurate, cyclohexane,
tetraethyl orthosilicate (TEOS), and potassium carbonate were
from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Silica
gel 60 N (spherical, neutral) was from Kanto Chemical Co. ING.
(Tokyo, Japan). Fluorescein sodium salt and fluorescein iso-
thiocyanate (FITC)-dextran (average MW: 20 000) were from
Sigma-Aldrich (St. Louis, MO, USA). Nile blue A was from Alfa
Aesar (Lancashire, UK).
Scheme 1 Preparation of the photocleavable monomer containing a BODIPY structure.
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(193 mg) and dibutyltin dilaurate (16 mg) were added to the Irradiation conditions
reaction with stirring at 75 1C for 48 h. The crude product was
A halogen lamp (LG-PS2; Olympus, Tokyo, Japan) was used as a
light source. The light was filtered with 500 nm band pass
filters (Edmund Optics Inc., Tokyo, Japan) and used to irradiate
the nanoparticle solution for 3 min at a power of 7 mW cmꢀ2
(at 500 nm).
purified by column chromatography over silica gel (n-hexane : ethyl
acetate = 3 : 1, 10% [w/w] potassium carbonate). The yield for the
photocleavable monomer was 59% (160 mg).
1H NMR (500 M Hz, CDCl3): d = 7.51 (t, aromatic–H), d = 7.09
(d, aromatic–H), d = 7.09 (d, aromatic–H), d = 6.62 (t, aromatic–H),
d = 5.87 (s, aromatic–H), d = 4.96 (s, aromatic–CH2O), d = 3.8
(m, Si(OCH2CH3)3), d = 3.18 (t, NHCH2CH2), d = 2.49 (s, aromatic–
CH3), d = 2.05 (s, aromatic–CH3), d = 1.62 (m, CH2CH2CH2), d = 1.25
(t, Si(OCH2CH3)3), d = 0.61 (t, CH2CH2Si).
Evaluation of the release efficiency of encapsulated molecules
from nanoparticles by visible light stimulation
After irradiation, the nanoparticle solution was concentrated by
ultracentrifugation (Optima TLX; Beckman Coulter) at 57 000 ꢁ
g for 10 min. The supernatant (100 ml) was poured into the wells
of a 96-well micro-assay plate (BD Biosciences, Franklin Lakes,
NJ, USA). The fluorescence intensity of the supernatant was
measured using a multiplate reader (SH-9000; Corona Electric
Co., Ibaraki, Japan). Excitation/emission wavelengths were
491/521 nm for fluorescein, 540/580 nm for rhodamine B,
and 635/675 nm for Nile blue A.
Preparation of visible light-responsive nanoparticles
Preparation of the seed and visible light-responsive nano-
particles. The seed and nanoparticles were prepared based on a
previously described method.20 Briefly, 9.1 mg of arginine were
dissolved in 6.9 ml of water, followed by the addition of 0.45 ml
of cyclohexane and 0.55 ml of TEOS in this order. The solution
was stirred at 60 1C for 20 h to obtain seeds of uniform size.
A solution of 50 ml of seed, 0.5 mg of arginine, 25 ml of ethyl
acetate, 20 ml of TEOS, 0.5 mg of photocleavable monomer, and
10 ml of encapsulated molecules (10 mg mlꢀ1 fluorescein sodium
salt, rhodamine B, Nile blue A, or FITC-dextran, 1 mg mlꢀ1 DAPI)
were added to 1 ml of water. The mixture was stirred overnight at
500 rpm and 60 1C. The prepared nanoparticles were purified by
dialysis (Spectra/Por 6, MW cutoff: 50 000; Spectrum Laboratories,
Inc., Compton, CA, USA) for 24 h to remove excess molecules that
were not encapsulated by the nanoparticles.
Cell culture
HeLa cells were cultured in a humidified atmosphere of 95%
air and 5% CO2 at 37 1C in Dulbecco’s Modified Eagle’s
Medium (DMEM, Sigma-Aldrich) supplemented with 1% (v/v)
penicillin–streptomycin solution (100ꢁ) (Wako Pure Chemical
Industries, Ltd) and 10% (v/v) foetal bovine serum (FBS; Invitrogen).
For each experiment, cells at 80–90% confluence were harvested by
digestion with trypsin/EDTA (Wako Pure Chemical Industries, Ltd),
then washed and resuspended in fresh growth medium with FBS at
a concentration of 106 viable cells/culture flask (75 cm2).
Surface modification of nanoparticles with R8
A 150 ml volume of nanoparticle solution and 150 ml of R8
peptide (4 mM in water) were mixed and vortexed for 20 min at
room temperature.
Internalisation of the nanoparticles by cells
Cells in the culture dish were washed with DMEM without
serum and 2 ml of DMEM were added. A 200 ml volume of as
prepared nanoparticle solution was added dropwise to the dish;
after allowing 1 h for internalisation, the dish was washed three
times with culture medium; after incubation, the internalisa-
tion of nanoparticles was evaluated from fluorescence images.
Transmission electron microscopy (TEM) measurements
TEM images were obtained using an H-7000 electron micro-
scope (Hitachi, Tokyo, Japan) operating at 75 kV. Copper grids
(400 mesh) were coated with a thin film of collodion followed by
carbon. The nanoparticle dispersion (5 ml) was placed on the coated
copper grids and dried at room temperature before observation.
Confocal laser scanning microscopy
Images of the cells were obtained using an LSM510 microscope
(Carl Zeiss AG, Oberkochen, Germany) using a W-PI 10ꢁ/23
eyepiece (Carl Zeiss AG) and a C-Apochromat 40ꢁ/1.2W UV-VIS-
NIR objective lens (Carl Zeiss AG) or using an Fv10i microscope
(Olympus).
Dynamic light scattering (DLS) measurements
A Nanotrac Wave DLS instrument (Microtrac BEL Corp., Osaka,
Japan) was used to measure the diameters of the nanoparticles.
Measurements were carried out at room temperature using a
780 nm laser beam. At least three replications were performed
for each sample. A sample solution of 20 ml was used for
measurement. Size distribution graphs of the relative intensity
of scattered light with respect to the hydrodynamic diameter of
nanoparticles were obtained.
Results and discussion
Visible light-responsive nanoparticle generation and
characterisation
We first prepared seed nanoparticles from tetraethyl orthosilicate
(TEOS) and modified their surface with a photocleavable
Zeta potential
The nanoparticle solution was diluted 10-fold with 10 mM NaCl monomer and TEOS to generate visible light-responsive nano-
solution prior to the measurement of zeta potential, which was particles (Fig. 1), which was similar to the procedure used to
carried out on a Delsa Nano C instrument (Beckman Coulter, prepare UV-responsive nanoparticles described in our previous
Inc., Brea, CA, USA).
report.9c
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functioned as glue. In contrast, in the case of UV-responsive
nanoparticles containing a nitrobenzyl moiety as the photo-
cleavable group,9c individual nanoparticles were observed to become
enlarged despite being prepared under the same conditions as
visible light-responsive nanoparticles. This difference may be
attributed to the hydrophobicity of the BODIPY structure, which
hindered the dispersion of nanoparticles in an aqueous solution.
The irradiation time for the release of encapsulated molecules
was set at 20 s using a light-emitting diode (60–100 mW at 350 nm)
for UV-responsive nanoparticles. Visible light-responsive nano-
particles were irradiated for a longer time (3 min) using a halogen
lamp (7 mW cmꢀ2 at 500 nm) for an irradiation strength that was
the same as that used in our previous study.9c Visible light
irradiation increased the average nanoparticle size to 300 nm as
determined by DLS, with more clusters observed by TEM analysis,
suggesting that irradiation caused alterations in the nanoparticle
surface properties that consequently accelerated cluster formation.
TEM results indicated that the grey colour of the structure between
bound nanoparticles became lighter by irradiation, which was
likely due to the cleavage reaction of the photoresponsive layer.
Since the size of each nanoparticle was similar to that of the seed
nanoparticle, the photoresponsive layer was presumed to form a
thin surface on the nanoparticle (Fig. 1).
Fig. 1 Schematic illustration of light-responsive nanoparticles and photo-
cleavage reaction of the BODIPY structure.
DLS measurements indicated that the average sizes of the
seed and functional nanoparticles were 25 and 70 nm, respec-
tively (Fig. 2a). A TEM analysis revealed that seed particles were
monodispersed and had globular form, whereas BODIPY nano-
particles formed clusters without significant changes in the
nanoparticle size (Fig. 2b). Thus, the increase in the nano-
particle size by modification with the cleavable monomer was
mainly due to clustering and not an increase in the size of
individual nanoparticles. In fact, the grey structures observed
between bound nanoparticles suggested that the monomer
Release and activation of encapsulated molecules by visible
light
Nanoparticles with fluorophores in the outer shell were prepared by
combining the photocleavable monomer TEOS and a fluorophore
in a dispersed solution of seed nanoparticles (Fig. 1). We compared
the fluorescence intensity of the supernatant before and after
irradiation to evaluate the amount of fluorophore released upon
visible light stimulation. Fluorescein, rhodamine B, and Nile blue A
were selected as representative fluorophores and irradiation time
was set to 3 min. Although photobleaching of organic fluorescent
dyes by long exposure times has been reported,21 the fluorescence
intensities of the three fluorophores were similar before and after
3 min (7 mW cmꢀ2) of irradiation (Table 1), indicating that this
exposure time did not induce photobleaching and confirming the
low risk of damage associated with blue-green light. In fact, the
fluorescence intensity of the supernatants of the three nanoparticle
solutions was increased by irradiation (Fig. 3), indicating that the
fluorophores were released as a result of changes to the nano-
particles induced by irradiation. Low fluorescence was observed in
the supernatants of the three unirradiated nanoparticle solutions,
which may have resulted from nanoparticles leaching during
ultracentrifugation (57 000 ꢁ g for 10 min). Only a slight increase
in fluorescence intensity was observed for the fluorescein-
containing nanoparticle, likely because it was not retained by
the negatively charged silica nanoparticle owing to electrostatic
Table 1 Ratio of fluorescence intensity before and after irradiation
Fluorophore
Fluorescein
Rhodamine B
Nile blue A
Fig. 2 (a) DLS and (b) TEM analyses of the seed nanoparticle (NP) and
visible light-responsive NPs before and after irradiation.
Ratio before and after
irradiation
104 ꢂ 2
100 ꢂ 1
97 ꢂ 2
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Fig. 3 Amount of fluorophore released from nanoparticles upon irradiation
with blue-green visible light.
repulsion. In contrast, the fluorescence intensities of the supernatant
of the rhodamine B- and Nile blue A-containing nanoparticles
increased by about 4- and 6-fold, respectively, upon irradiation. This
was similar to the increase observed for the UV-responsive nano-
particles that we previously investigated.9c There was no observable
difference in the release properties, although the monomer cleavage
group was changed from a nitrobenzyl to a BODIPY group.
Fig. 4 Confocal laser scanning micrographs of HeLa cells treated with
(a) rhodamine B, (b) rhodamine B-containing nanoparticles, (c) R8-modified
rhodamine B-containing nanoparticles, (d) FITC-dextran, (e) FITC-dextran-
containing nanoparticles, and (f) R8-modified FITC-dextran-containing
nanoparticles. (g) Comparison of fluorescence intensity of cells treated with
fluorophore and nanoparticles.
Cell internalisation of visible light-responsive nanoparticles
We investigated whether the nanoparticle could be internalised
by cells and thus serve as a molecular carrier. Rhodamine B and
FITC-dextran were used as the encapsulated fluorophores.
Fluorophore only, fluorophore-containing nanoparticles, and
octaarginine (R8)-modified fluorophore-containing nanoparticles
were added to HeLa cell culture dishes, and after washing, the
fluorescence intensity of the cells was measured (Fig. 4). The
fluorescence intensity of the each solution was aligned. R8 is a
cell-penetrating peptide (CPP) that enhances the permeation of the
cell membrane.22 There was weak fluorescence detected in cells
treated with rhodamine B and none in cells treated with FITC-
dextran, indicating that the latter does not penetrate the cell
membrane. However, fluorescence was observed in cells treated
with fluorophore-containing nanoparticles, both with and without
internalisation. The change in zeta potential to a positive value
also confirmed the modification of the nanoparticle surface with
R8, although this did not significantly improve internalisation
efficiency. These results indicate that the light-responsive nano-
particles are an effective vehicle for the delivery of cargo mole-
cules into cells.
Spatiotemporal release and activation of cargo molecules
within cells by visible light
R8. This suggests that the nanoparticles facilitated the internalisa- To determine whether the nanoparticles can function as photo-
tion of the fluorophore. There was no obvious difference between activatable carriers, we examined the release of cargo molecules
nanoparticles with and without CPP modification (Fig. 4d). Thus, within cells upon irradiation. DAPI was chosen as a cargo
the developed nanoparticles can be taken up by cells without molecule, since its fluorescence intensity is increased about
requiring surface modification.
20-fold by complex formation with DNA.23 There was almost
To examine the mechanistic basis for the internalisation, the zeta no fluorescence observed in cells that internalised DAPI-
potential of the nanoparticles was determined. The zeta potentials of containing nanoparticles (Fig. 5a). This is because the DAPI
the seed, unmodified, and R8-modified nanoparticles were ꢀ14, ꢀ1, molecule was encapsulated within the nanoparticle mesh
and 7 mV, respectively. Although the seed was originally negatively structure, which hindered the interactions with DNA. In con-
charged, the charge was effectively neutralized by modification with trast, strong fluorescence was observed throughout the cells
the photocleavable monomer, and became positive by additional upon irradiation for 3 min (7 mW cmꢀ2, Fig. 5b and d),
modification with R8. The polarity change of the nanoparticles’ presumably because encapsulated DAPI was released and
zeta potential likely reduced electrostatic repulsion from the formed complexes with DNA. This indicated that internalised
negatively charged cellular membrane thereby accelerating their nanoparticles were not exocytosed by cells, but instead escaped
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and Technology (MEXT) of Japan and JSPS Core-to-Core Program,
A. Advanced Research Networks.
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such as a CPP and were induced to release cargo molecules by
irradiation with visible light. Although various light-responsive
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