PH-Responsive near-infrared fluorescent cyanine dyes for molecular imaging based on pH sensing

Article abstract of DOI:10.1039/c7cc03035e

Indocyanine green (ICG) derivatives having nucleophilic substituents were synthesized as pH-responsive near-infrared dyes. pH-responsive dyes 1-C with closed-ring structures smoothly internalized and converted to emissive open-ring structures 1-O in respo

Full text of DOI:10.1039/c7cc03035e

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pH-Responsive Near-Infrared Fluorescent Cyanine Dyes for
Molecular Imaging Based on pH Sensing
Koji Miki,*a Kentaro Kojima,a Kazuaki Oride,a Hiroshi Harada,b Akiyo Morinibu,b and Kouichi Ohe*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Indocyanine green (ICG) derivatives having nucleophilic low-contrast images. Cy7 dyes like ICG, having proton-
substituents were synthesized as a pH-responsive near-infrared accepting groups on the -backbone, have been developed as
π
dye. pH-Responsive dyes 1-C with closed-ring structures smoothly pH-responsive probe molecules, but their pH-responsive
internalized and converted to emissive open-ring structures 1-O in ranges are difficult to tune (Figure 1b).7,9 Because the main
response to relatively low pH in acidic intracellular compartments fluorophore of ICG is composed of the π-conjugated push–pull
of HeLa cells.
tetraene structure having an electron-deficient iminium group,
one assumes that protic nucleophiles can react with an
electron-deficient iminium carbon atom to afford non-emissive
dyes under high-pH conditions. Raymo and co-workers
reported photoresponsive and/or pH-responsive cyanine dyes
with a nucleophilic 4-nitrophenol-2-yl moiety for sensor
Dyes that emit strong fluorescence through photoirradiation
are one of the most powerful probes for visualizing target
tissues by both in vitro and in vivo optical imaging devices.1,2
For in vivo use, there are two large problems in utilizing probe
molecules: (1) autofluorescence from normal tissues and (2)
low contrast because of background signal from the probes in
normal tissues or blood vessels. To overcome the former
problem, researchers focused their attention on near-infrared
m
a
t
e
r
i
a
l
s
,
(d) labeling agents (known compounds )
(a) indocyanine green (ICG)
dyes, which can work in the optical window3 (
λ = 700–900 nm)
N
N
3
N
N
to reduce autofluorescence. One of the answers to the latter
problem is stimuli-responsive dyes.1g,4 Stimuli-responsive
dyes, especially responding to pH change, have been
intensively investigated for applying high-contrast visualization
of living cells in vitro and tissues in vivo. It is known that the
pH values around inflamed sites and tumour tissues are nearly
6.0 or even lower, sensitively detected by pH-responsive
probes.5 Although excellent pH-responsive visible dyes with
xanthene and boron–dipyrromethene structures for visualizing
tumour tissues have been reported,6 there are few examples
of pH-responsive near-infrared dyes for in vivo use.5a,7
3
n
n
Z
Z
-O3S
4
SO3Na
4
H
H
n = 2 or 3
labeling agents
Z = NH or O
H+
(b) pH-responsive Cy7 derivatives (known compounds )
A1
A1
H+
A2
N
N
N
R
N
R
3
H
H+
(A1, A2 = proton acceptor)
(c) pH-responsive ICG derivatives (this work)
HX
N
N
Indocyanine green (ICG, Figure 1a), which was approved to be
used for diagnosis of cardiac output and hepatic function, is
one of the best-known near-infrared fluorescent
heptamethine cyanine (Cy7) dyes.8 Because of its non-stimuli-
responsive property, ICG can visualize not only target tissues
but also normal tissues and blood vessels, therefore providing
3
N
3
HX
X-
N
Et
Y
n
Et
n
YH
1-O (open-ring structure, fluorescent)
1a 1b
1-C (closed-ring structure, non-fluorescent)
1c ,
(Y = NH, n = 5)
(Y = NH, n = 1),
(Y = NH, n = 2),
1d (Y = O, n = 2), 1e (Y = S, n = 2), 1f (Y = COOH, n = 2)
Figure 1. (a) Commercially available indocyanine green (ICG)
and (b) known pH-responsive Cy7 derivatives. (c) pH-
Responsive ICG derivatives 1. Equilibrium between 1-O having
an open-ring structure and 1-C having a closed-ring structure.
(d) Cy7 derivatives known as labelling agents.
although almost all reported dyes work in the visible region.10
In this context, we have developed ICG derivatives having a
nucleophilic subunit, such as aminopropyl, hydroxypropyl, and
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mercaptopropyl groups, on a benzoindole nitrogen atom as a of an amino group of another dye 1c to form a dimer under
highly and precisely tunable pH-responsive near-infrared dye, high pH conditions. From the UV–vis absorption
and have evaluated their pH-responsiveness in buffered measurements of 1d and 1e, both 1d-C and 1e-C smoothly
solutions and in a biological environment (Figure 1c). The converted to the open-ring structure 1d-O and 1e-O under
annulation of intramolecular nucleophilic moieties smoothly slightly acidic conditions (Figures 2d and 2e). The pH-
proceeded to afford non-emissive dyes 1-C having a closed- responsive ranges of 1d and 1e were observed around pH 5–7
ring structure under high-pH conditions. Under low-pH and pH 3–6, respectively (Figure 3a). Their pH-responsiveness
conditions, in contrast, heteroatoms in the cyclic structures of was also confirmed by tracing the change of fluorescence
1-C were smoothly protonated to form 1-O having an open- regarding 1d-O and 1e-O in various buffered aqueous
ring structure, leading to the recovery of near-infrared solutions, although their pH-responsive ranges slightly shifted
fluorescence emission even in living cells. Cy7 derivatives to higher pH values (Figure 3b). The difference in pH-
having aminopropyl, hydroxypropyl and hydroxyethyl groups responsive ranges of 1b, 1d and 1e would be due to the
on a benzoindole nitrogen atom have been reported as difference in the basicity of cyclic amine, ether and sulfide.
fluorescent labelling agents, but their pH responsiveness has The drastic spectroscopic change of the UV–vis spectrum of 1f
not referred to the function of nucleophilic moieties (Figure having a carboxy group was not observed even under strongly
1d).11 Because it is known that the pH of individual cellular basic conditions, probably because of the low nucleophilicity
organelles and compartments in cells are different,12 we have of the carboxylate ion (Figure 2f). The reversibility of pH
carefully investigated the pH responsiveness of the newly responsiveness was evaluated while the pH values of a
developed 1-C
/
1-O in vitro.
buffered solution of 1e between pH 3 and 7 were changed
ICG derivatives
1
having nucleophilic substituents—amino, several times. The repeatable on/off switching of absorbance
hydroxy, mercapto and carboxy groups—were synthesized at 800 nm and fluorescence at 820 nm were observed without
according to our reported synthetic procedure of 1c (see the significant change (Figure 3c).
Electronic Supplementary Information (ESI)).13 To evaluate the
(a)
(c)
(e)
(b)
(d)
(f)
pH responsiveness, UV–vis absorption spectra of
1 in buffered
0.4
0.2
0
0.15
aqueous solutions were measured. When the absorption
spectrum of an aqueous solution (3.0 mL, pH 7.4) containing
1a in dimethyl sulfoxide (1.5×10-3 M, 10 μL) and Triton X-100
(2.0×10-3 M) as a surfactant was measured, strong absorption
of an ICG fluorophore at 780 nm was detected along with a
signal around 400–500 nm (Figure 2a). From a time-
dependent density functional theory calculation, the broad
absorbance around 400–500 nm was assigned to 1a-C having a
closed-ring structure (see the ESI). By measuring buffered
aqueous solutions of 1a-C under various pH conditions, the
pH-dependent change of absorption intensities was recorded.
As the pH values of solutions decreased from 11 to 7, the
absorption signal of 1a-C decreased and the corresponding
signal of 1a-O increased. The absorbance of the ICG
fluorophore was detected at pH 11.4, indicating that 1a-O
cannot fully convert to 1a-C, even under strongly basic
conditions. An absorption signal attributed to J-aggregates14
was also detected around 850–900 nm in all the solutions
because 1-O undergoes facile aggregation in water. By
measuring the absorbance of aqueous solutions without Triton
X-100, we observed that the aggregation could be promoted
irrespective of the pH values (see the ESI). The isosbestic point
low
low
0.1
pH
pH
high
high
0.05
0
300
300
500
700
900
500
700
900
wavelength (nm)
wavelength (nm)
0.6
0.3
0
0.8
0.4
0
low
low
pH
pH
high
high
300
500
700
900
300
500
700
900
wavelength (nm)
wavelength (nm)
0.6
0.3
0
0.8
0.4
0
low
pH
high
could not be clearly detected in solutions of 1a-O/1a-C
with/without Triton X-100, probably because dye aggregation
could not be suppressed even in the presence of a surfactant.
In the case of 1b, the pH-responsive range slightly shifted to
higher pH values, probably because 5-endo-trig cyclization is
less favourable than 6-endo-trig cyclization according to
Baldwin’s rules (Figure 2b).15 In the case of 1c, the absorption
intensity at 780 nm slightly decreased as pH values of buffered
aqueous solutions increased (Figure 2c). Because 9-endo-trig
cyclization is less favourable, we assumed that an iminium
moiety of 1c underwent the intermolecular addition reaction
Figure 2. UV–vis absorption spectra of buffered aqueous
solutions of dyes (5.0
10–6 M), (a) 1a (pH = 11.2, 9.4, 7.4, 5.7),
×
(b) 1b (pH = 11.4, 9.4, 8.0, 7.4, 5.8), (c) 1c (pH = 11.4, 9.4, 7.4,
5.7), (d) 1d (pH = 9.5, 7.4, 6.5, 5.8, 5.0), (e) 1e (pH = 7.4, 5.8,
5.0, 4.0, 2.5) and (f) 1f (pH = 2.8, 7.4, 11.4), with triton X-100
(2.0
×
10–3 M).
2 | J. Name., 2012, 00, 1-3
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(a)
(b)
fluorescence, while no fluorescence was detected in the case
of 1e at 4 °C. We supposed that ICG having a positively
charged iminium nitrogen atom could interact with cell
membranes consisting of negatively charged phospholipids
even at 4 °C. We consider that positively charged 1d-O among
1d-C and 1d-O in a neutral solution would similarly bind to cell
membranes. Because the fluorescence of cells treated with 1d
at 37 °C was twice as strong as that treated with ICG, 1d-C
having a neutral hydrophobic closed-ring structure efficiently
internalized into cells through endocytosis and transformed to
1.0
0.8
0.6
0.4
1.0
0.8
0.6
0.4
0.2
0.0
0.2
0.0
2
4
6
8
10
12
2
4
6
8
10
12
pH
pH
(c)
(d)
1.0
0.8
0.6
0.4
1.0
0.8
0.6
0.4
emissive 1d-O. Although 1e-O is a minor component in a
weakly acidic aqueous solution of 1e, the large F37/F4 of 1e
clearly suggests that 1e can visualize acidic tissues with high
contrast.
0.2
0.0
0.2
0.0
7
3
7
3
7
3
7
3
7
3
7
7
3
7
3
7
3
7
3
7
3
7
pH
pH
Figure 3 (a) UV–vis absorbance of 1b (triangle), 1d (circle) and
1e (cross) in buffered aqueous solutions normalized at 780 nm,
and (b) Fluorescence intensities of them normalized at 820 nm
(λex = 780 nm). pH-Dependent change of (c) absorbance (Abs)
and (d) fluorescence (FL) of 1e. Abs and FL were normalized by
those measured at pH 3.0.
To evaluate the pH responsiveness of dyes in living cells, we
next incubated HeLa cells with ICG, 1d and 1e at 4 °C (on ice,
to prevent endocytosis) or 37 °C for 4 h in Dulbecco’s modified
Eagle’s medium (DMEM) containing foetal bovine serum (FBS,
10%). The commercially available ICG exhibited the same
fluorescence intensities regardless of culture temperature
(Figures 4a-c). In contrast, the fluorescence intensities of 1d
and 1e gradually but markedly increased at 37 °C but not at 4
°C. Similar data were obtained using 1e at a concentration of
Figure 4. Fluorescence intensity ratios (F/F’) of HeLa cells
×
10–4 M, indicating that endocytosis was not affected by
incubated in DMEM solutions (2.0 mL) with ICG (blue, 1.0
5 M), 1d (green, 1.0 10–5 M) or 1e (red, 1.0 10–5 M) at (a) 4
°C and (b) 37 °C. F4, F37: fluorescence intensities of cells
incubated at °C and 37 °C, respectively. F’4, F’37:
fluorescence intensities at 4 °C and 37 °C just after treatment
(0 h), respectively. (c) Representative false colour
×
10–
1.0
dye concentration. There is a statistically significant difference
in fluorescence intensity between the incubation at 37 °C and
4 °C (p < 0.05). This indicates that 1d-C and 1e-C were
gradually internalized into cultured cells through the ATP-
dependent endocytosis and were converted to 1d-O and 1e-O
(open-ring structures) by responding to the acidic intracellular
compartments of HeLa cells, respectively. The fluorescence
×
×
4
fluorescence images and (d) average fluorescence intensities
of HeLa cells incubated for 4 h in DMEM solutions (2.0 mL)
with ICG (1.0
1d (1.0
10–5 M) at 4 °C and 37 °C. Green and orange bars:
× × ×
10–5 M), 1e (1.0 10–4 M), 1e (1.0 10–5 M) or
increment of 1d at 37 °C was four times higher than that of 1e
,
suggesting that treatment with 1d can afford brighter images
under acidic conditions in cells and visualize small tissues
clearly. However, by considering that stronger fluorescence
×
incubated at 4 °C and 37 °C, respectively. *: p<0.1, **: p<0.05.
was detected from cells just after treatment with 1d, 1e is
In summary, we have developed ICG derivatives having
nucleophilic subunits, such as amino, hydroxy and mercapto
groups, as a pH-responsive near-infrared fluorescent dye.
These dyes showed pH-dependent equilibrium between a
fluorescent open-ring structure and a non-fluorescent closed-
ring structure in the ranges of pH 7–9, pH 5–7 and pH 3–6,
respectively. From in vitro cell culture experiments using HeLa
cells, both 1d and 1e were found to show the pH-dependent
enhancement of fluorescence intensively through endocytosis.
The control incubation experiments at 4 °C clarified that
positively charged pH-responsive dyes having an open-ring
more suitable for detection of tissues as well as cell activities
such as endocytosis without background noise signal. After
washing cells with phosphate buffered saline, to evaluate the
fluorescence intensities of probes specifically in HeLa cells, the
ratio (F37/F4) of fluorescence intensities from cells incubated
with ICG, 1d or 1e at 37 °C (F37) to those at 4 °C (F4) were 2.3,
4.3 and 28, respectively. These results also support our
conclusion that dyes having closed-ring structures were
smoothly converted to their emissive forms after
internalization through the ATP-dependent endocytosis.
Interestingly, cells treated with ICG and 1d at 4 °C emitted
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15 Because J-aggregate of 1b-O was observed in UV-vis
absorption spectra under acidic conditions, the fluorescence
intensities of 1b-O was overestimated under basic conditions
(see the ESI).
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C7CC03035E
Journal Name
COMMUNICATION
Graphical abstract
pH-Responsive near-infrared cyanine dyes were synthesized and
applied as an imaging probe of acidic intracellular compartments
of live cells.
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