Photoreversible cellular imaging using photochrome-conjugated fullerene silica nanoparticles

Article abstract of DOI:10.1039/c1cc14041h

Photochromic compound-conjugated fluorescent fullerene-silica nanoparticles prepared by the reverse-microemulsion method was utilized for photoswitchable cellular imaging by repeatable irradiation of ultraviolet and visible light.

Full text of DOI:10.1039/c1cc14041h

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COMMUNICATION
Photoreversible cellular imaging using photochrome-conjugated fullerene
silica nanoparticlesw
Jinyoung Jeong,a Eunju Yun,a Yohan Choi,a Hye-youn Jung,a Sang J. Chung,a
Nam Woong Songb and Bong Hyun Chung*a
Received 6th July 2011, Accepted 25th August 2011
DOI: 10.1039/c1cc14041h
Photochromic compound-conjugated fluorescent fullerene-silica
nanoparticles prepared by the reverse-microemulsion method
was utilized for photoswitchable cellular imaging by repeatable
irradiation of ultraviolet and visible light.
state upon UV irradiation and an open-ring form upon visible
light irradiation, by either breaking or forming a chemical
bond.12 On the basis of these characteristics, we designed a
photoswitchable nanoparticle by combining a dithienylethene
derivatised photochromic compound (PC) and a highly bio-
compatible, photoluminescent fullerene-silica nanoparticle
(FSNP), which were previously developed and utilised as
photoreversible bioimaging agents with low autofluorescence
in a variety of cells.13
Fluorescent nanoparticles have been extensively investigated
for biomedical applications such as bioimaging because of their
unique properties, which include strong photoluminescence,
tunable colour based on size, and tolerance for cell penetration.1,2
Semiconducting quantum dots, fluorescent dye-encapsulated
silica nanoparticles, and fluorescent carbogenic nanoparticles
are widely used for cell imaging, monitoring of target molecules
in tissue, and detection of biological interactions in vivo.3,4
However, there remain several challenges, including high
background autofluorescence from biomolecules, photo-blinking,
and high cytotoxicity.5 Recently, photoswitchable nanoparticles
have been studied as a means to overcome these drawbacks in
bioimaging applications. In particular, polymer nanoparticles
containing fluorescent dyes and spiropyran were used for dual-
colour imaging of complex biological systems based on fluores-
cence resonance energy transfer (FRET).6–8 Although the
FRET-based imaging system enables precise, specific and
multiplex imaging of target molecules, there are considerable
drawbacks, such as the need to design acceptor and donor
molecules, to maintain the proper distance between molecules,
and to make further modifications for cell penetration.
In this study, we synthesised an amine-functionalised
dithienylethene that can directly conjugate to fullerene via
nucleophilic addition by the following procedure (see Fig. S1,
ESIw). The photochrome-conjugated fullerene silica nano-
particles (PC-FSNPs) were prepared by addition of ammonium
hydroxide (NH4OH) and the PC solution to a reverse micro-
emulsion containing the C60 fullerene and silica precursor
(TEOS) (Fig. 1). Because the amine group of the PC was very
attracted to the C60 fullerene, it was necessary to add the
NH4OH to the microemulsion with the PC to induce simulta-
neous cross-linking of the silica and fullerene via hydrolysis of
TEOS and addition of the amine to C60.
The size and shape of the as-prepared PC-FSNP was measured
by scanning electron microscopy (SEM). As seen in Fig. 2,
the PC-FSNP was highly monodisperse with an average
diameter of 85.22 Æ 5.85 nm. It was slightly larger than the
previously prepared FSNP (61.5 Æ 6.0 nm), indicating that the
conjugation of the PC into the nanoparticle may affect its size.
Photochromism occurs when a chemical species has two
forms that are reversibly transformed by irradiation at different
wavelengths. This phototransformation phenomenon can be
affected by a variety of physicochemical properties, including
the refractive index, dielectric constant, and geometric structure.
Dithienylethene is a promising photochromic compound because
of its thermal irreversibility and resistance to fatigue.9–11 It can
be reversibly switched between two conformations: a closed-ring
a BioNanotechnology Research Center, Korea Research Institute of
Bioscience and Biotechnology, Gwahangno, Yuseong,
Daejeon 305-806, Republic of Korea. E-mail: chungbh@kribb.re.kr;
Fax: +82 42 879 8594; Tel: +82 42 860 4442
b Cell Dynamics Research Center at GIST and Nanobio Fusion
Research Center, Korea Research Institute of Standards and Science,
Daejon 305-340, South Korea
w Electronic supplementary information (ESI) available: Experimental
details and supplementary data. See DOI: 10.1039/c1cc14041h
Fig. 1 Schematic illustration of synthesis of photochrome-conjugated
fullerene-silica nanoparticles via the reverse microemulsion method.
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Fig. 2 FE-SEM image of as-prepared PC-FSNP. In the inset, the
photograph shows the colour change of the PC-FSNP aqueous
solution under UV and visible light irradiation.
The nanoparticles were light yellow under white light due to
the structure-broken fullerene; however, they clearly became
greenish-yellow after UV irradiation (Fig. 2, inset), due to the
mix with blue colour of the closed-ring state of PC. On the
contrary, the nanoparticle prepared without C60 did not
change the colour upon either UV or visible light illumination
(data not shown), indicating that PC did not interact with the
silica precursor.
To characterise the chemical composition of the PC-FSNP,
Fourier-transform infrared (FTIR) spectroscopy was conducted.
In the FTIR spectrum of the PC-FSNP, the Si–O signal from
1100 cmÀ1 to 1300 cmÀ1 was too strong to indicate whether
the covalent C–N bond between C60 and amine-functionalised
dithienylethene was present. Therefore, the PC-FSNP was
treated with sodium hydroxide to eliminate silica, and an FTIR
spectrum was obtained. Fig. S2 (ESIw) shows the C–N absorption
in the range of 1250 cmÀ1 to 1350 cmÀ1, indicating the formation
of an aromatic nitrogen–carbon bond. It demonstrates that
amine-terminated PC was covalently conjugated to C60
via nucleophilic attraction.14 It is consistent with the result
obtained from the IR spectrum of the conjugate of PC
and C60 which shows the aromatic carbon–nitrogen bond at
1261 cmÀ1 (inset in Fig. S2, ESIw). In addition, the C–F
Fig. 3 Optical properties of PC-FSNP in aqueous solution under UV
and visible light irradiation. (a) UV/visible absorption spectra of
PC-FSNP (inset: UV/visible spectra of photochrome in ethanol) and
(b) Photoluminescence spectra of PC-FSNP (inset: photoluminescence
spectra of FSNP).
region of the PC matched the photoluminescence (PL) wave-
length (B600 nm) of the FSNP, we expected that the PL of the
PC-FSNP was influenced by the change in absorption due to
the phototransformation of the PC. To confirm this hypothesis,
we measured the PL of the PC-FSNP and FSNP control using
an Ar ion laser (excitation at 488 nm). The resulting PL
spectrum of the PC-FSNP under visible light was similar to
that of the FSNP, indicating that the conjugation of the PC
did not affect the luminescence of the FSNP, which originates
from the C60–O–Si bond. However, the PL intensity decreased
about 52% after UV light irradiation but recovered after
visible light irradiation. The dependency of the PL on the
wavelength used was assumed to be caused by the emissive
energy of the FSNP being transferred to the PC, as it strongly
absorbs visible light at 600 nm after UV irradiation. In other
words, the PL of the PC-FSNP was quenched by the inter-
molecular energy transfer between the C60–O–Si and the PC in
the nanoparticle after UV irradiation.15 After the ring structure
of dithienylethene was transformed back by visible light
irradiation, the PL also recovered to its original intensity, in
clear contrast to the FSNP, which had no change in PL whether
irradiated with either UV or visible light (Fig. 3b, inset). The
quenching effect can be influenced by various factors, such as
the molar ratio of donor and acceptor molecules in the system,
the distance between the two molecules, and the surrounding
elements. Although it is complicated to distinguish the exact
effect the different wavelengths used had on the quenching
efficiency of PC-FSNP, it is roughly determined by the number of
PC molecules in a single nanoparticle, calculated from weight loss
analysis in TGA, and the distance from the dithienylethene
vibration peaks corresponding to the PC at 951.6 cmÀ1
,
1056.4 cmÀ1, and 1383.5 cmÀ1 were also observed. Thermo-
gravimetric analysis (TGA) of the particles shows a weight
loss at 512 1C, corresponding to the elimination of the silanol
(Si–OH), that was about 20% less for the PC-FSNP than the
FSNP control, which implies the PC was conjugated to the
nanoparticles (see Fig. S3, ESIw). This conclusion assumes that
the PC competes with silica to conjugate to the C60 during the
particle formation. Taking the above results into account, we
believe that the nanoparticles were formed by the covalent
immobilisation of the PC and silica onto C60 via nucleophilic
substitution and silanisation, respectively.
The optical properties of PC-FSNP in aqueous solution
were also investigated under both UV and visible light irradiation.
As seen in Fig. 3a (inset), the PC had an absorption peak at
600 nm under visible light that increased after UV (365 nm)
irradiation as a result of the transformation to the closed-ring
state. The absorbance recovered upon irradiating with visible
light (512 nm). Although the change in the absorption of the
PC-FSNP was relatively small, the spectra were similar to
those of the pure PC under sequential UV and visible light
irradiation, whereas the FSNP control had no change in the
absorption spectra (data not shown). Because the absorption
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and the FSNP after UV irradiation. The intensity from the
PC-FSNP decreased considerably compared to that of the
FSNP, which had no major changes in fluorescence under either
UV or visible light. Furthermore, the intensity was reversibly
changed by repeatedly irradiating with UV and visible light up
to 8 times (Fig. 4b). The average intensity difference between
UV and visible light irradiation was 48.1 Æ 1.7%, which is
similar to the quenching efficiency of PC-FSNP (52%) in an
aqueous solution. It is noteworthy that the photoreversible
reaction of the PC-FSNP occurred consistently in both the
pristine solution and the intracellular environment.
In conclusion, we prepared photoreversibly switchable
photoluminescent nanoparticles through the conjugation of
an amine-functionalised photochrome, C60, and silica by the
reverse microemulsion method. The PL of the nanoparticles
was reversibly and repeatedly switched on and off upon UV
and visible light irradiation through the intermolecular energy
transfer between the photo-induced transformation of PC and
C60–O–Si, the PL species. The quenching efficiency (about 50%)
of PC-FSNP by UV irradiation in both a pristine solution and
an intracellular environment was comparable to that of other
photoreversible switching systems. This nanosystem consisted
of a photoswitchable molecule and a rigid, photostable and
biocompatible photoluminescent moiety could be used for
photoreversible analysis during cellular imaging and detection
of target molecules in a complex biological system with a high
signal-to-noise ratio.
Fig. 4 (a) Fluorescence images of PC-FSNP-containing HeLa cells.
The images were taken under a combination of 490 nm excitation/617 nm
emission filter after UV and visible light irradiation for 5 min each.
(b) Comparison of fluorescence intensity of PC-FSNP and FSNP
under repeatable UV and visible light irradiation.
moiety to the C60 through the tetraethyleneglycol linker, which
is close enough for energy transfer to occur (o5 nm). Compared
to the quenching efficiency of FRET and other systems,
PC-FSNP is a reasonable system for photoreversible analysis
or imaging owing to its rigid structure surrounding the photo-
transformation moiety, its high photostability, and the bio-
compatibility originating from the FSNP.
The authors acknowledge the financial support received from
the Pioneer Research Center Program (Grant No. 2008-00180,
MEST, Korea), the Fundamental R&D Program for Core
Technology (MKE, Korea), and the KRIBB Initiative Program
(KRIBB).
Notes and references
In light of these advantages, we evaluated the photoreversible
cellular imaging of PC-FSNP uptake in cells with sequential
UV and visible light irradiation. Before the cellular imaging, we
investigated the time-dependent quenching effects of PC-FSNP
to determine an adequate irradiation time. The relative fluores-
cence intensity of the PC-FSNP in an aqueous solution was
measured every minute after either UV or visible light irradiation.
As seen in Fig. S4 (ESIw), the intensities after UV irradiation
gradually decreased by almost 50%, while the intensities after
visible irradiation increased within 5 min. After 5 min, there
were no further changes in either the quenching or recovery of
the PL of the PC-FSNP. Therefore, the irradiation time for the
photoreversible effect of the PC-FSNP was fixed at 5 min.
For cellular imaging, the HeLa cells were incubated with
PC-FSNP and FSNP (50 mg mlÀ1 for each) for cellular uptake
by endocytosis. Next, the cells were washed with the media
and PBS after one-hour incubation. The fluorescent images of
the nanoparticle-containing cells were obtained by fluores-
cence microscopy with an emission filter of 617 nm under
excitation at 488 nm. As seen in Fig. 4a, both the PC-FSNP-
and FSNP-containing cells showed bright red emission in the
cytosol region of the cells after visible light irradiation,
indicating that both the PC-FSNPs and FSNPs were signifi-
cantly internalised into the cells. However, there was a distin-
guishable difference in the red fluorescence of the PC-FSNP
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This journal is The Royal Society of Chemistry 2011