Cell imaging using red fluorescent light-up probes based on an environment-sensitive fluorogen with intramolecular charge transfer characteristics

Article abstract of DOI:10.1039/c4cc04654d

We report a general strategy to design and synthesize red fluorescent light-up probes for cellular imaging based on a fluorogen with intramolecular charge transfer characteristics. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc04654d

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Cell imaging using red fluorescent light-up probes
based on an environment-sensitive fluorogen with
intramolecular charge transfer characteristics†
Cite this: Chem. Commun., 2014,
0, 9497
5
Received 19th June 2014,
Accepted 3rd July 2014
ab
a
a
ac
Guangxue Feng,‡ Jie Liu,‡ Ruoyu Zhang and Bin Liu*
DOI: 10.1039/c4cc04654d
www.rsc.org/chemcomm
We report a general strategy to design and synthesize red fluor- When the fluorophore and the quencher are located within a
escent light-up probes for cellular imaging based on a fluorogen specific distance, the fluorescence of the probe is quenched. Once
with intramolecular charge transfer characteristics.
they are separated by specific biological events, the fluorescence of
the probe could be recovered. Another example is a singly labelled
Fluorescence imaging has become an indispensable tool in the probe, which typically contains a fluorescence silent signal subunit
study of living systems due to its high sensitivity, low cost, that responds to an analyte or links to a binding unit/recognition
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multiple parameters (e.g., intensity, wavelength and lifetime) as site. The molecular recognition process is often accompanied by
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well as activatable or multiplexed signals. However, the photo- fluorescence recovery. For example, some silent fluorophores can
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limiting interferences in biological media, such as autofluores- recognize target analytes, such as enzymes, metal ions or signal-
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cence, tissue absorption and scattering, limit fluorescence detec- ling molecules, in biology to turn on the fluorescence. Probes
tion from being widely applied in practical settings. If the probes containing fluorogens with aggregation-induced emission (AIE)
could be engineered to remain fluorescence silent and only be characteristics also belong to this category. These probes are
switched on in recognition of a specific biological event, it would non-fluorescent when molecularly dissolved, but become highly
provide a large signal-to-noise ratio with minimized background fluorescent once aggregates are formed or upon restriction of
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fluorescence. On the other hand, employment of long wavelength intramolecular rotation induced by a biological event. In
fluorescent probes is also an alternative to reduce the photo- addition, few probes containing environment sensitive fluoro-
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limiting interference due to the minimal interferential absorption phores have also been developed, which have weak fluores-
and high tissue penetration ability. As a result, it is of high cence in polar media, and the fluorescence light-up occurs when
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importance to develop smart fluorescent probes possessing both the probe is bound to hydrophobic protein domains. So far, the
long wavelength emission and the ability to significantly enhance AIE fluorogens and the environment sensitive dyes used for
their fluorescence in response to biological stimuli.
light-up probe design are limited to those with blue or green
To date, several strategies have been employed to design fluor- emission. It is highly desirable to develop specific fluorescent
escent light-up probes for cellular imaging. One is to cage the light-up probes with long wavelength emission for cell imaging.
fluorescent dye with photochemical labile groups, where the fluores-
One efficient way to design long wavelength emissive fluor-
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cence is generated upon UV light irradiation. The other example is ophores is the combination of electron rich and electron
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dual labelled probes containing a fluorophore–quencher pair.
deficient units to form a donor (D)–acceptor (A) structure.
The obtained molecules often possess intramolecular charge
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transfer (ICT) characteristics, and their fluorescence is sensi-
a
Department of Chemical and Biomolecular Engineering, National University of
Singapore, Singapore 117576, Singapore. E-mail: cheliub@nus.edu.sg;
Fax: +65-6779-1936
tive to the polarity of the environments, e.g., lower fluorescence
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in high polar media. One could expect that these molecules
b
c
can be designed to have very low fluorescence in a highly polar
environment (e.g., aqueous environment), while the fluores-
cence is enhanced when the environment becomes hydropho-
bic or less polar. In addition, our previous studies have shown
that conjugated polyelectrolytes with ICT characteristics have
enhanced fluorescence upon aggregation due to their reduced
Environmental Research Institute, National University of Singapore, 117411,
Singapore
Institute of Materials Research and Engineering, 3 Research Link, 117602,
Singapore
†
Electronic supplementary information (ESI) available: Experimental details,
Lippert plot, titration and DFT calculation of DBT, NMR, EIMS, HPLC, LLS and
photostability of DBT-KRRRQRRKKR, the CLSM image of EDBT treated cells. See
DOI: 10.1039/c4cc04654d
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exposure to polar environments. These examples further
inspire us to develop a new strategy to design and synthesize
‡
G. Feng and J. Liu contributed equally to this work.
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Fig. 1 (A) UV-vis and (B) PL spectra of EDBT in hexane, toluene, tetra-
hydrofuran, dichloromethane, acetonitrile and dimethyl sulfoxide. [EDBT] =
2
mM. The chemical structure of EDBT is shown in the inset of (A).
Scheme 1 (A) Chemical structure of DBT-KRRRQRRKKR; (B) schematic
illustration of the switching mechanism for the fluorescent light-up probe.
environment sensitive light-up probes by taking advantage of
ICT characteristics. In this contribution, we selected thiophene
as the electron donor and highly electron-deficient 2,1,3- reaction. The detailed synthetic route and procedures are
benzothiadiazole as an electron acceptor to construct a donor– provided in Scheme S1 (ESI†). The chemical structures of key
acceptor–donor (D–A–D) structured fluorophore. A water soluble intermediates and the final probe were characterized by
1
peptide was further incorporated into one side of the fluoro-
H NMR, EIMS and HPLC (Fig. S4–S7, ESI†). The probe shows
phore to impart the probe with good water solubility. After cell good solubility in aqueous media. The switching mechanism
uptake, the fluorescence of the probe is significantly enhanced. of the probe is proposed in Scheme 1B. When the probe is
We first studied the ICT characteristics of 4-(5-ethynylthio- molecularly dissolved in aqueous media, its fluorescence is very
phen-2-yl)-7-(thiophen-2-yl)-2,1,3-benzothiadiazole (EDBT, the weak due to strong ICT-induced fluorescence quenching. When
chemical structure is shown in the inset of Fig. 1A), by measuring the probe interacts with cell components or proteins after cell
its absorption and emission spectra in solvents with different uptake, the increase in environment hydrophobicity will light-
polarities (dielectric constant (e)). As shown in Fig. 1A, all the up the fluorescence.
absorption spectra have maxima at B453 nm, and there are no
The UV-vis absorption spectrum of DBT-KRRRQRRKKR was
obvious changes observed in all six solvents. Along with an recorded in 150 mM phosphate buffered saline (PBS). As shown
increase of e, the emission maxima, however, progressively red- in Fig. 2A, DBT-KRRRQRRKKR has two absorption peaks
shift from 542 nm in hexane (e = 2.0), to 563 nm in toluene centred at 325 and 462 nm, which arise as a result of a p–p*
(e = 2.4), 578 nm in tetrahydrofuran (THF, e = 7.5), 583 nm in transition centred on the donor units along with the charge
dichloromethane (DCM, e = 9.1), 587 nm in acetonitrile (ACN, transfer between the donor (thiophene) and the acceptor (ben-
e = 36.6) and 597 nm in dimethyl sulfoxide (DMSO, e = 47.2), zothiadiazole) units, respectively. The red-shifted absorption
concomitant with a gradual decrease in the fluorescence intensity maximum of DBT-KRRRQRRKKR relative to EDBT reveals that
15
(
Fig. 1B). The Lippert–Mataga plot (Fig. S1 in the ESI†) shows the triazole ring formed after the click reaction also contributes
that EDBT has small Stokes shifts in aprotic solvents (e.g. hexane) to the effective conjugation length. As shown in Fig. 2B, the PL
with low orientational polarizability, but with largely increased maxima of DBT-KRRRQRRKKR show clear red-shifts from
À1
Stokes shifts over 5000 cm in polar solvents (e.g. DMSO). These 609 nm in ACN to 621 nm in DMSO and 650 nm in 150 mM
results reveal that EDBT is an environment-polarity-sensitive PBS buffer (e = 80.1), accompanied with a decrease in the
fluorophore. Moreover, the fluorescence of EDBT can be effec- fluorescence intensity, indicating that the probe is indeed
tively quenched in water to yield very low fluorescence (Fig. S2, sensitive to the environment polarity.
ESI†), which provides us an opportunity to develop fluorescent
To evaluate the capability of DBT-KRRRQRRKKR as a fluor-
light-up probes by taking advantage of the environment-polarity escent light-up probe, the titration experiments were carried
changes. The density functional theory (DFT) calculation of EDBT out in the cell medium upon addition of cell lysate. The PL
was also performed by using a suite of Gaussian 03 program. The spectra were obtained upon excitation at 462 nm. DBT-
lowest unoccupied molecular orbital (LUMO) of EDBT is domi- KRRRQRRKKR solution shows very weak fluorescence in the
nated by the orbitals from the benzothiadiazole moiety, while cell medium with an emission maximum at B650 nm. Upon
the electron clouds of the highest occupied molecular orbital gradual addition of cell cytoplasmic lysate, the solutions exhibit
(HOMO) are mainly located on both thiophene and benzothia- enhanced bright red fluorescence, accompanied with a blue-
diazole units (Fig. S3, ESI†). The difference in the electron cloud shift of emission maxima from B650 nm to 616 nm (Fig. 2C).
distribution shows intrinsic ICT properties, which is consistent The gradual blue-shift of emission maxima indicates that the
with the fluorescence titration results.
microenvironment of the probe becomes relatively more hydro-
We subsequently designed and synthesized the probe, phobic, which is beneficial to suppress charge transfer-induced
DBT-KRRRQRRKKR, and the chemical structure is shown in fluorescence quenching. The fluorescence change is also asso-
Scheme 1A. A cell penetrating peptide KRRRQRRKKR was ciated with aggregate formation, which is evidenced by the
conjugated with EDBT to afford DBT-KRRRQRRKKR via a click laser light scattering. The hydrodynamic size of aggregates
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Fig. 3 CLSM images of living MCF-7 cancer cells treated with DBT-
Fig. 2 (A) UV-vis spectrum of DBT-KRRRQRRKKR in 150 mM PBS solution.
KRRRQRRKKR at different concentrations for 1, 2, 3, and 4 h, respectively.
(
B) PL spectra of DBT-KRRRQRRKKR in ACN, DMSO, and 150 mM PBS
À1
The nuclei were pre-stained with nucleus staining dye Hoechst (5 mg mL
)
solution. (C) PL spectra of DBT-KRRRQRRKKR in the presence of the cell
for 20 min. All the images share the same scale bar.
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lysate based on cell density ranging from 0 to 2.2 Â 10 cells per mL. (D)
0
Titration experiments depicted by I/I as a function of cell lysate based on
cell density. I is the maximum fluorescence intensity in the presence of cells,
and I is the initial maximum fluorescence intensity of the probe alone.
DBT-KRRRQRRKKR] = 2 mM.
light up in the nucleus. The results coincide well with the
literature on penetration peptide mediated cellular uptake, which
occurs at two stages: binding to the membrane followed by
disintegration of the membrane and internalization into the
0
[
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formed between DBT-KRRRQRRKKR and the cell lysate cytoplasm. The fluorescence light-up in the nucleus is due to
increases from B1 nm to more than 1 mm when the cell lysate the nucleus localization signalling function of the peptide.
5
based on the cell density is increased from 0 to 2.2 Â 10 cells
To study the mechanism of fluorescence light-up in cells, we
per mL (Fig. S8, ESI†). It is worth noting that the maximum also titrated DBT-KRRRQRRKKR with bovine serum albumin
fluorescence intensity increases by B32-fold relative to the (BSA) or lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine
probe alone in the absence of cell cytoplasmic lysate (DPPC)) (Fig. S12, ESI†). The probe showed similar fluorescence
(Fig. 2D), indicating that the probe has a large signal-to-noise enhancement as that by titration with the cell lysate. Moreover,
ratio, which is crucial for fluorescent light-up probes. The light- the probe showed instant fluorescence light-up upon mixing
up probe exhibits almost no fluorescence change in the cell with the cell lysate, and the fluorescence remained almost
lysate when the pH is varied from 5 to 8 (Fig. S9, ESI†), revealing unchanged for 4 h. As the peptide hydrolysis takes time, the
the insensitivity of DBT-KRRRQRRKKR to intracellular pH.
results indicate that the light-up mechanism should be mainly
DBT-KRRRQRRKKR is subsequently used as a fluorescent due to the interaction between the probe and cellular proteins
light-up probe for live cell imaging. MCF-7 breast cancer cells or the membrane, which provides a hydrophobic microenvir-
were chosen as a model cell line for the fluorescence imaging onment for probe fluorescence enhancement. These results
study by confocal laser scanning microscopy (CLSM). After suggest that DBT-KRRRQRRKKR can serve as a potential cell
incubation with 2 mM EDBT or DBT-KRRRQRRKKR at 37 1C uptake-mediated fluorescent light-up probe for cell imaging.
for 0, 1, 2, 3, and 4 h, respectively, the images were obtained
Photostability comparisons among DBT-KRRRQRRKKR,
upon excitation at 488 nm with red fluorescence signals Alexa fluor 488 and fluorescein were evaluated under contin-
collected above 560 nm. Almost no fluorescence is observed for uous laser scanning upon illumination at 488 nm with a power
MCF-7 cells upon incubation with EDBT alone (Fig. S10, ESI†), of 1.25 mW. Only B12% fluorescence intensity loss was
indicating that peptide conjugation is essential for cell uptake. observed for DBT-KRRRQRRKKR after 10 min illumination,
On the other hand, when the images were immediately taken which is better than Alexa fluor 488 (B23% signal loss) and
after the cells were incubated with 2 mM DBT-KRRRQRRKKR fluorescein (B100% signal loss) (Fig. S13, ESI†), suggesting
(0 h), no fluorescence was observed (Fig. S11, ESI†), indicating that DBT-KRRRQRRKKR has good photostability. In addition,
that the probe exhibits very weak fluorescence in the culture the cytotoxicity results evaluated by MTT assays reveal that the
medium outside the cell. As shown in Fig. 3, some fluorescence cell viabilities still remain above 80% after 48 h of incubation
on the cell membrane is observed after 1 h incubation. As the with a 5 mM probe (Fig. S14, ESI†).
incubation time gradually increases to 4 h, the bright red fluores-
In conclusion, we used a new strategy to design and synthe-
cence is gradually spread through the whole cytoplasm even in the size a cell uptake-mediated red fluorescent light-up probe for
nucleus (Fig. 3). We also varied the DBT-KRRRQRRKKR concen- cell imaging. The fluorophore has a D–A–D structure, which
trations for the cellular imaging. At lower concentration (0.5 mM), allows us to take advantage of the ICT characteristics to light up
the probe tends to stay longer on the cell membrane, while higher its fluorescence by changing the environment polarity from
probe concentration (10 mM) benefits fast cell uptake and quick polar to non-polar (e.g. upon cellular uptake and aggregate
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formation). This work provides a general design principle to
develop red fluorescent light-up probes for cellular imaging
without washing steps. In the future, we will use such design
principles to develop NIR fluorescent light-up probes with
targeting ability through conjugation with specific ligands for
targeted cellular imaging.
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9500 | Chem. Commun., 2014, 50, 9497--9500
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