Amphiphilic diarylethene as a photoswitchable probe for imaging living cells

Article abstract of DOI:10.1021/ja8043163

This communication reports a unique example of water-soluble and fluorescent-switchable amphiphilic diarylethene. This compound performs stable vesicle aggregation in water and shows aggregation-dependent emission in its open form. The fluorescence can be effectively switched by alternating between UV and visible light irradiation. This compound thus can stain KB cells for switchable living cell imaging with excellent resistance to fatigue. Copyright

Full text of DOI:10.1021/ja8043163

Published on Web 10/31/2008
Amphiphilic Diarylethene as a Photoswitchable Probe for Imaging Living Cells
Ying Zou, Tao Yi,* Shuzhang Xiao, Fuyou Li, Chunyan Li, Xia Gao, Junchen Wu, Mengxiao Yu, and
Chunhui Huang
Department of Chemistry and Laboratory of AdVanced Materials, Fudan UniVersity, Shanghai 200433, P.R. China
Received June 7, 2008; E-mail: yitao@fudan.edu.cn
Scheme 1. Structure and Photochromic Process of 1
Fueled by advancements in imaging technology, examination of
cells by fluorescence techniques has been widely used to study
cellular processes.1,2 Conventional fluorescent probes for fluorescent
imaging, such as rhodamine3 and cyanine dyes,4 are useful for
cellular studies but can respond only irreversibly to one event.5 In
contrast, fluorophores that can respond reversibly provide a simple
and powerful technique for regional optical marking and thus would
be much more valuable for cellular fluorescent labels.6 Though
several photoactivatable fluorescent proteins have been developed
to make possible precise photo labeling and tracking of the target
protein,6c the large size of fluorescent proteins often results in
improper protein functioning. Therefore, the development of a novel
small photocontrollable fluorescent labeling molecule would be a
powerful tool to elucidate the protein dynamics in living cells.7
Very recently, Li et al. reported a dual-color fluorescent probe
based on spiropyran loaded nanoparticles;6b nevertheless, the
irradiation source provides shorter than 365 nm UV light and the
thermal instability of the open-ring form of spiropyran limited its
application in bioimages. It is well-known that diarylethene is the
most promising switchable unit within those classical photochromic
systems owing to its excellent fatigue resistance and thermal
stability.8 However, most of these studies are carried out in organic
systems, including the newly developed fluorescent photochromic
diarylethene for labeling biomolecules by M. Irie,7 which is not
suitable for biological application. These problems may be resolved
by inducing amphiphilic molecules, which is a widely used
technique to construct highly organized nanostructures in aqueous
systems with the driving force of hydrophobic interaction.9 Herein
an amphiphilic molecule (1, Scheme 1) was designed with
hydrophilic and hydrophobic chains at two ends of a rigid
diarylethene core. 1 forms stable vesicles in water and exhibits
obviously different fluorescence in open and closed states under
alternating irradiation of UV and visible light. Thus, this molecule
would serve well as a photoswitchable probe for imaging living
cells.
were obviously blue-shifted (40 nm) with enhanced intensity when
the concentration increased from 2.5 × 10-6 to 1.0 × 10-5
M
(Figure 2A). However, in regular organic solvents such as methanol,
the fluorescence was completely quenched for the breaking of
aggregation (Figure S2, SI) and became intense and blue-shifted
as we added water. This finding determines the strongly aggrega-
tion-dependent fluorescence of 1 in water. The confocal laser
scanning microscopy (CLSM) images for one drop of 1O water
solution (1.0 × 10-5 M) on a glass substrate showed well-dispersed
micrometer-sized fluorescent circles (Figure 1C), indicating that
the hydrophobic interaction and the hydrophilic repulsion between
the molecules governed the arrangement of the molecules to
vesicles.9 Those vesicles can disperse in aqueous solution and
remain unchanged for more than a month in a concentration range
The formation of the aggregate state of 1 in aqueous solution
was proved by the plot of the conductivity of 1 (open isomer, 1O)
as a function of the concentration with the critical aggregation
concentration at ∼1.2 × 10-6 M (Supporting Information, SI). A
scanning electron microscope (SEM) image shows a spherical
morphology with a diameter of 50-200 nm for the freeze-dried
sample from a low-concentration aqueous solution (1.0 × 10-5
M, Figure 1A). On the other hand, column-shaped single crystals
and aggregated crystals were obtained from the mixed solvent of
water and methanol 1:1 (v/v, 1.0 × 10-5 M) (Figure 1B).
Similar to the previously studied diarylethene pyridinium moi-
eties,10 1O exhibited greenish fluorescent emission centered at 510
nm in water (1.0 × 10-5 M), with the fluorescent quantum yield
(QY) being 0.0044 by using Rhodamine B as reference. A slightly
increased QY was found with increasing concentration (For
example, QY ) 0.0073 for 5.0 × 10-5 M of 1O). The emissions
Figure 1. SEM images of 1O (A) from water solution (scale bar, 0.5 µm);
(B) from the mixed solvent of H2O/MeOH ) 1:1 (v/v) (scale bar, 2 µm).
CLSM images of 1O (C) in water (1.0 × 10-5 M); (D) in mixed solvent
of H2O/MeOH ) 1:5 (v/v, 1.0 × 10-5 M); (E) in PBS solution (2.0 ×
10-5 M). λex ) 405 nm, λem ) 480-580 nm.
Figure 2. (A) Concentration-dependent fluorescent emission spectra of 1O
(a: 1.0 × 10-5 M, b: 7.5 × 10-6 M, c: 5.0 × 10-6 M, d: 2.5 × 10-6 M in
water). (B) Absorption and (C) emission spectral changes of 1O under 365
nm light irradiation (1.0 × 10-5 M, λex ) 385 nm).
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10.1021/ja8043163 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
blue staining cell viability experiment gave a cell viability of more
than 99.5 (%) incubated with 20 µM 1 for 20 min, which shows
that 1 has low cytotoxicity. A selected cell was then irradiated with
a 405 nm light for 3 min, and the fluorescence of the irradiated
cell was turned off (Figure 3A2), with the denotation of 1 in the
cells changing from an open state to a closed state. All cells can
also be lamped off upon 405 nm irradiation for 3 min (Figure 3A3).
It is amazing that the fluorescence of the cells could be recovered
by 633 nm light irradiation from the CLSM lamp for 1 h (Figure
3A4).
The reversibility and stability of the cell images by the utility of
1 was demonstrated in fixed KB cells. The microfluorescence
switching of fixed KB cells was achieved while alternating between
UV and visible light illumination. We were excited that, as evident
in Figure 3B and Figures S9, S10, the optical switching of
fluorescence can be repeated many times without any apparent
“fatigue” effects, or photobleaching, that were thought to be the
fateful disadvantage of the general fluorophore. Thus, we concluded
that 1 was a novel and superior fluorescence dye for use as a cell
marker or fluorescence switch in a cell system.
In conclusion, we developed a new amphiphilic diarylethene,
which forms stable vesicle nanostructures in aqueous solution and
exhibits switchable fluorescence between open and closed states.
These fluorescent vesicles can enter the living cells with low
cytotoxicity and have potential utility as a cellular marker or
fluorescence switch in living cells with high-ratio signal change
and excellent resistance to fatigue. We expect that such materials
will be of great benefit to biomedical researchers for further study.
Figure 3. (A) CLSM image (above) and the overlay image (bottom) of
KB living cells incubated with 1O for 20 min at 25 °C (1) in original state,
(2) irradiated by 405 nm light (2 mW) for a single cell, (3) all cells, and
(4) recovered by 633 nm light (0.7 mW) (λex ) 405 nm, 0.15 mW). (B)
Fluorescence switching of fixed KB cells by alternating UV (405 nm, 2
mW, 10 s/time) and visible (633 nm, 0.7 mW, 20 min/time) light
illumination. Inset: images of one cycle.
of 1.2 × 10-6-7.5 × 10-5 M. With addition of methanol to the
aqueous solution, the vesicles changed to entangled fibers (Figure
1D).
Acknowledgment. We thank NSFC (20771027, 20571016,
20490210),SSTC(08JC1402400,06PJ14016),NHTPC(2006AA03Z318),
and SLADP (B108) for financial support.
1O has a maximum absorption at 385 nm (extinction coefficient,
ꢀ385 ) 6700 L ·mol-1 ·cm-1) and performs obvious photochromism
in aqueous solution. Irradiation of colorless 1O with 365 nm light
led to a new absorption at 640 nm with a photocyclic quantum
yield of 33.3% (Figure 2B), accompanied by quenching of most of
the fluorescence (98% quenched within 30 s in photo stationary
state [PSS]) because of the enlargement of π-electron delocalization
(Figure 2C).8 Under visible light irradiation, both absorption and
fluorescence were completely converted into the open isomer
according to the spectral change, showing the classical switchable
property of diarylethene. This water-“soluble” fluorescent switchable
material thus provides a nice platform for application in bioimaging
owing to the high-ratio signal change in water.
With CLSM, we then developed a practical application of 1 as
a fluorescence switch in a biological system (Figures S4, S5). After
KB cells (human nasopharyngeal epidermal carcinoma cell) were
incubated with a PBS solution (pH ) 7) of 20 µM 1O (0.6-1.0
µm in size from DLS and CLMS, Figures 1E, S3) for 20 min at 25
°C, we observed a marked fluorescence increase in the cytoplasm
of the cells (Figure 3A1). Intense intracellular luminescence with
a high signal-to-noise ratio (I1/I2 > 50) was detected between the
cytoplasm and background (Figure S6). Further quantization by line
plots revealed large signal ratios (I1/I3 )14) between the cytoplasm
and nucleus, implying that weak nuclear uptake occurred for 1.
The luminescence was still clearly observed when the KB cell was
loaded with 1 at 4 °C, indicating that 1 is unlikely to enter the
cells by endocytosis (Figure S7), which may be beneficial for
intracellular target recognition. The cytotoxic characteristic is
important for 1 as a bioprobe; therefore, the effect of 1 on cell
proliferation was determined by means of an MTT assay (SI). The
cellular viabilities were estimated to be greater than 85% in 24 h
in the presence of 0.2-20 µM 1 (Figure S8). Meanwhile, a Trypan
Supporting Information Available: Synthetic and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
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