Y. Gong et al.
Dyes and Pigments xxx (xxxx) xxx
dry DMF (5 mL), the mixture was stirred at 50 ◦C. After the reaction was
completed (monitored by TLC, about 2–3 h), the mixture was diluted
with CH2Cl2, washed with water for 3 times. The organic phase was
dried over with Na2SO4, and purified by silica column chromatography
using CH2Cl2/CH3OH (100:1 to 20:1, v/v) as eluent to afford MTR-HH
optical in vivo imaging system. All live cells and live animal operations
were in accord with institutional animal use and care regulations, ac-
cording to protocol No. SYXK (Xiang) 2008–0001, approved by Labo-
ratory Animal Center of Hunan.
1
as a purplish red solid in granules or sheets (248 mg, yield 68%). H
2.9. Measurement of fluorescence quantum yields
NMR (CD3OD + DMSO‑d6, 400 MHz) δ (ppm): 8.28 (d, J = 14.4 Hz, 1H),
8.11 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.70–7.67 (m, 3H),
7.56 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.4 Hz,
1H), 7.12–7.09 (m, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 14.8 Hz,
1H), 5.19 (s, 2H), 4.67 (q, 2H), 2.62–2.58 (m, 4H), 1.80–1.77 (m, 2H),
1.38 (t, J = 7.2 Hz, 3H), 1.28 (s, 12H). 13C NMR (CD3OD + DMSO‑d6,
100 MHz) δ (ppm): 169.9, 161.2, 157.9, 154.0, 142.7, 141.3, 139.9,
134.9, 130.5, 129.3, 128.7, 127.5, 127.3, 127.2, 127.0, 127.0, 123.9,
115.8, 115.6, 113.2, 112.5, 105.3, 101.7, 84.0, 70.1, 43.5, 28.9, 24.8,
24.5, 20.4, 13.5. HRMS (ESI) calculated for C37H39BNO4S+ [M ꢀ Iꢀ ]
604.2687, found 604.2667.
Fluorescence quantum yields of MTR and MTR-HH were determined
by using indocyanine green (ICG, ΦF = 0.13 in DMSO) as fluorescence
standards. The quantum yield was calculated using the following
equation:
Φ
F(X) ⋅ = ⋅ ΦF(S) ⋅ (ASFX ⋅ / ⋅ AXFS)⋅(nX /nS)2
where ΦF is the fluorescence quantum yield, A is the absorbance at the
excitation wavelength, F is the area under the corrected emission curve,
and n is the refractive index of the solvents used. Subscripts s and x refer
to the standard and to the unknown, respectively. The solutions of
compounds MTR, MTR-HH and ICG were kept absorptions below 0.05.
2.7. Cytotoxicity study
3. Results and discussion
Cytotoxicity study was performed using the CellTiter 96 Aqueous
One Solution cell proliferation assay (MTS) (Promega, USA)). HeLa cells
(2 × 105) were seeded in 30 mm glass bottomed dishes and incubated for
48 h. After removing cell medium, cells were first incubated with MTR
3.1. Design motivation and synthesis
Though two works have been reported for the benzothiazole
substituted hemi-cyanine, the authors have to bear some drawbacks
including low overall yield and difficult to treatment [37,38]. Herein,
benzothiazolecyanine dye (2-[2-[2-chloro-3-[2-(3-ethyl-3H-benzothia
zol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3-ethylbenzo-
thiazol-3-ium iodide) was facilely synthesized according to the mature
steps. Then the present improved preparation of hemi-benzothia
zolecyanine dye (2-[2-(2,3-dihydro-6-hydroxy-1H-xanthen-4-yl)ethe-
nyl]-3-ethylbenzothiazol-3-ium iodide, MTR) between resorcin and
benzothiazolecyanine was carried out with high yield. And MTR reacted
with 4-bromomethylphenylboronic acid pinacol ester to produce the
final probe MTR-HH. Specific synthesis and characterization data are
listed in supporting information.
and MTR-HH (2, 4, 6, 8 and 10 μM, respectively) in 1 mL Dulbecco’s
Phosphate-Buffered Saline (Dꢀ PBS, 1.47 mM KH2PO4, 8.06 mM
◦
Na2HPO4, 137.93 mM NaCl, 2.67 mM KCl) at 37 C for 2 h to allow
sufficient uptake. Afterwards, supernatant was removed from the test
well following centrifugation, and 200 μL of fresh cell culture medium
were added. After another 48 h of incubation, a 6 × diluted MTS solution
(120 μL/well) in DMEM medium solution was added to each well and
incubated at 37 ◦C for 2 h. The absorbance value at 490 nm was
determined by a microplate reader.
2.8. Cell incubation and fluorescence imaging in vivo
Living HeLa cells and RAW 264.7 cells were obtained from the
Biomedical Engineering Center of Hunan University (Changsha, China)
and cultured in DMEM (Dulbecco’s modified Eagle’s medium) supple-
3.2. Photophysical properties of dye MTR
◦
mented with 10% FBS (fetal bovine serum) at 37 C in a 5% CO2 at-
mosphere. For dose-dependent imaging of exogenous H2O2, HeLa cells
were washed with PBS buffer (pH 7.4) 3 times, four groups were treated
We first investigated the photophysical properties of hemi-
benzothiazolecyanine MTR in various solvents and pH conditions. As
shown in Fig. 1, owing to the hydroxyl group, MTR exhibits obvious
solvation effect of organic solvents and PBS (pH 7.4, 40 mM PBS, 5%
MeOH as a co-solvent) with two main absorption bands at about 600
nm and 680 nm, while the dye shows bathochromic absorption bands
at 735 nm in strong polar solvents DMSO and DMF, resulting in
different distinctive colors in different solvents by naked eyes. Though
complete emission peaks of MTR in all solvents could be obtained
when excited at 600 nm, they are irregular which are composed of
maximum peak and shoulder peak. In contrary, emission peaks of MTR
in only PBS, DMSO and DMF are located in range 700–900 nm when
excited at 680 nm, accordingly, the following fluorescence tests were
carried out with excitation wavelength of 680 nm. In sum, photo-
physical properties including maximum absorption wavelengths,
molar absorption coefficients, fluorescence emission wavelengths and
fluorescence quantum yields of MTR and the present probe MTR-HH
are listed in Table S1. In Fig. 1d–f, as the enhancement of pH from 3.0
to 11.0, the main absorption band at 600 nm undergo a bathochromic-
shift to a new peak at 701 nm, attribute to deprotonation process of
MTR during pH changing. Meanwhile, pH-dependent fluorescence
profiles of MTR is consistent with that of the absorption, the
enhancement of pH induces a turn-on fluorescence signal with a pKa
value of MTR calculated to be 5.8. In addition, MTR was treated with
with 0, 20, 50 and 100 μM H2O2 for 30 min, respectively. Each group
was then incubated with the probe MTR-HH (5 μM) for another 60 min.
For imaging of endogenous H2O2, RAW 264.7 cells were divided into
three groups. The first group works as the control experiment without
addition in the medium; the second group was first incubated with
phorbol myristate acetate (PMA, 2.0
bation with MTR-HH (5.0
first incubated with phorbol myristate acetate (PMA, 2.0
acetyl-L-cysteine (NAC) for 2 h, followed by incubation with MTR-HH
(5.0 M) for another 60 min. For photostability experiments, HeLa cells
stained with MTR (5.0 M) for 30 min, were imaged after laser illumi-
nation for 0, 5, 10, 15 and 20 min. For co-localization experiments, the
cells treated with probe MTR (5.0 M) and Mito-Tracker Red CMXRos
(0.2
μ
g/mL) for 2 h, followed by incu-
M) for another 60 min; the third group was
g/mL) and N-
μ
μ
μ
μ
μ
μ
M) for 30 min. All cells were washed three times with PBS, and the
fluorescence images were then performed through an Olympus FV1000-
IX81 confocal fluorescence microscope with excitation at 543 nm for
green channel (580–650 nm), excitation at 635 nm for red channel
(690–750 nm).
Four-week-old male BALB/c nude mice only given intraperitoneal
injection of MTR-HH (5 μM, 20 μL) for 60 min was assigned as the
control group. Mice given an intraperitoneal injection of lipopolysac-
charide (LPS, 50 μM, 20 μL) for 4 h, followed by MTR-HH (5 μM, 20 μL)
for another 60 min were assigned as the positive group. Fluorescence
imaging was observed under a Caliper IVIS Lumina XR small animal
10–50
μ
M biological reductant (H2S), nucleophilic reagent (HSOꢀ3 ) and
oxidants (H2O2, O2⋅–, ⋅OH, HClO, NO, ONOOꢀ ) for 30 min, as shown in
4