Halogenated cyanine dyes for synergistic photodynamic and photothermal therapy

Article abstract of DOI:10.1016/j.dyepig.2021.109327

Cyanine dyes are widely used in the field of tumor phototherapy due to their excellent photophysical properties. To explore the heavy atoms effects on the photothermal and photodynamic performance of phototherapeutic agents, chlorine, bromine, and iodine were introduced to synthesize a series of cyanine dyes (IR6, IR7, and IR8). We have found that all halogenated cyanine dyes exhibited high excitation wavelength (around 800 nm) and low dark toxicity. Among them, IR8 behaved the best singlet oxygen production ability in the three dyes. For photothermal performance, IR8 exhibited the best photothermal conversion rate (46.6%), photothermal stability, and excellent therapy efficiency (half-maximal inhibitory concentration, IC₅₀ = 16.2 μg/mL). IR7 behaved a greater enhancement of the photothermal conversion rate (43.4%) than IR6 (42.3%). In conclusion, the heavy atoms effects on the photothermal and photodynamic properties of cyanine dyes are positively correlated with the increase of the atomic number of the halogen atom, and the iodine atom may be the most worthy of consideration in the all halogen atoms.

Full text of DOI:10.1016/j.dyepig.2021.109327

Dyes and Pigments 190 (2021) 109327
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Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Halogenated cyanine dyes for synergistic photodynamic and
photothermal therapy
Hao Liu, Juanjuan Yin, Enyun Xing, Yingying Du, Yu Su, Yaqing Feng, Shuxian Meng*
School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin, 300050, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cyanine dyes are widely used in the field of tumor phototherapy due to their excellent photophysical properties.
To explore the heavy atoms effects on the photothermal and photodynamic performance of phototherapeutic
agents, chlorine, bromine, and iodine were introduced to synthesize a series of cyanine dyes (IR6, IR7, and IR8).
We have found that all halogenated cyanine dyes exhibited high excitation wavelength (around 800 nm) and low
dark toxicity. Among them, IR8 behaved the best singlet oxygen production ability in the three dyes. For pho-
tothermal performance, IR8 exhibited the best photothermal conversion rate (46.6%), photothermal stability,
Cyanine dye
Photodynamic therapy
Photothermal therapy
Phototheranostic
Heavy atom effect
and excellent therapy efficiency (half-maximal inhibitory concentration, IC50 = 16.2 μg/mL). IR7 behaved a
greater enhancement of the photothermal conversion rate (43.4%) than IR6 (42.3%). In conclusion, the heavy
atoms effects on the photothermal and photodynamic properties of cyanine dyes are positively correlated with
the increase of the atomic number of the halogen atom, and the iodine atom may be the most worthy of
consideration in the all halogen atoms.
1. Introduction
from exerting effective photodynamic effects [11]. How to overcome the
defects of PTT and PDT and achieve better tumor treatment effects, has
Cancer is the leading cause of consistently high morbidity and
mortality worldwide [1]. Traditional cancer treatments include
chemotherapy, radiation therapy, and surgical treatment. However,
these treatments always make patients suffer from serious side effects
and drug resistance [2,3]. As one of the new treatments for cancer,
phototherapy has attracted widespread attention from researchers due
to its excellent properties such as non-invasiveness, high tumor sup-
pression, and high safety in tumor treatment [4]. Phototherapy mainly
includes photothermal therapy (PTT) and photodynamic therapy (PDT)
[5]. Among them, PTT is a method that uses photothermal agents (PTAs)
to convert light energy into thermal energy to generate high tempera-
ture and kill cancer cells under laser irradiation [6]. However, the
photothermal effect will be affected by the uneven distribution of PTAs
in tumors, leading to inefficient treatment [7]. Besides, the heat resis-
tance caused by the overexpression of heat shock proteins in some
cancers also makes the treatment less effective [8]. For PDT, reactive
oxygen species (ROS), especially singlet oxygen (1O2), produced by
photosensitizers (PSs) under laser irradiation can induce cell apoptosis
[9,10]. However, the hypoxic microenvironment at the tumor site limits
the production of reactive oxygen species and therefore prevents PSs
been an important target for researchers. And various new strategies
combining different tumor treatment methods have been developed. For
example, synergistic therapy, which combines PDT and PTT, can use the
photothermal conversion capacity of phototherapy agents to generate
heat to kill most tumor cells avoiding being influenced by the hypoxic
environment. Besides, the PSs can produce more ROS to kill tumor cells
with higher temperature achieving the effect of “1 + 1 > 2” [12,13].
Therefore, it is a particularly important method to develop phototherapy
agents that have excellent photothermal and photodynamic therapeutic
effects.
Indocyanine green (ICG), is the only Food and Drug Administration
(FDA) approved NIR agent [14]. It has also been explored as a potential
photosensitizer, but the disadvantages of photobleaching and low
singlet oxygen quantum yield have limited its practical application [15].
Therefore, a new kind of indocyanine green derivatives (Cypate [16],
IR780 [17], IR808 [18], IR825 [19], etc.) have been synthesized as
photosensitizers and photothermal agents. Among them, a heptame-
thine, IR-808 [20], was developed not only with NIR imaging properties
and good biocompatibility but also with photosensitizing activities [18,
21]. However, the therapeutic efficacy of IR-808, as well as many other
* Corresponding author. School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300050, PR China.
E-mail address: msxmail@tju.edu.cn (S. Meng).
https://doi.org/10.1016/j.dyepig.2021.109327
Received 29 November 2020; Received in revised form 29 January 2021; Accepted 14 March 2021
Available online 27 March 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

H. Liu et al.
Dyes and Pigments 190 (2021) 109327
Fig. 1. (a) Structures of halogenated cyanine dyes IR6, IR7, and IR8. (b) Synthesis procedure for IR6.
Fig. 2. (a) UV–Vis absorption spectra of IR6, IR7, and IR8 in methanol, (b) Fluorescence spectra of IR6, IR7, and IR8 in methanol, (c) UV–Vis absorption spectra of
IR6, IR7, and IR8 in water, (d) UV–Vis absorption spectra of IR6, IR7, and IR8 in DMF.
cyanine dyes, was limited by their inherent poor water solubility and
poor photostability, as well as the low quantum yield of singlet oxygen
production [22]. Therefore, to develop more excellent photothermal
agents and photosensitizers, it is important to investigate the influences
of substituent groups on the photothermal and photodynamic effects of
cyanine dyes.
the intersystem crossing (ISC), which can produce ROS [23]. For PTT, it
also involves electrons in excited triplet state and excited singlet state,
which are converted back to the ground state by nonradiative transition
and generate heat to kill tumor cells [24]. The effect of heavy atom has a
very important influence on the formation of triplet excited states.
In this paper, to investigate the influence of heavy atoms on photo-
physical properties, photothermal and photodynamic effects of cyanine
dyes, a series of halogenated cyanine dyes have been synthesized by
introducing the chlorine, bromine, and iodine into the structure of
heptamethine dye (IR6, IR7, and IR8), respectively. It came to the
conclusion that the effect of heavy atoms depending on the relationship
For phototherapy, all phototherapy agents would occur energy
conversion or transfer under light excitation. In PDT, singlet oxygen is
produced from energy transition from the sensitizers excited triplet state
to the ground state of molecular oxygen. Generally, it is involved a
process that excited singlet states transform into excited triplet states by
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Dyes and Pigments 190 (2021) 109327
Fig. 3. The absorbance of DPBF with IR6, IR7, and IR8 in (a) water and (b) DMF under laser in 10 min.
Fig. 4. (a) Temperature elevation for IR6, IR7, and IR8 (40
μ
g/mL) in water (10% DMF) under the irradiation of 808 nm laser at 1.0 W/cm2. Photothermal stability
experiments with alternate heating and cooling cycles of (b) IR6, (c) IR7, and (d) IR8 using an 808 nm laser at 1.0 W/cm2.
with the absorption/emission wavelength, photothermal conversion
efficiency, and photothermal stability was positively correlated with the
increase of the atomic number of the halogen atom. However, the in-
crease of the atomic number of halogen atom did not show a correlation
with the increase in the photodynamic effect. Moreover, IR8 exhibited
various advantages of an ideal PS with high photothermal conversion
rate, enhanced ROS production ability, low dark toxicity, and
photocytotoxicity.
purchased from Aladdin (Shanghai, China). All the raw chemicals were
purchased from commercial sources and used without further purifica-
tion. 1,3-diphenylisobenzofuran (DPBF) and 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Beyotime
Biotechnology. Dulbecco’s modified Eagle medium (DMEM), penicillin/
streptomycin fetal bovine serum (FBS), phosphate buffered saline (PBS)
were obtained from Baibei Biotechnology Co., Ltd. (Suzhou, China).
1H NMR and 13C NMR spectra were recorded on a Bruker Avance
400 MHz. High-resolution mass spectra (HRMS) were recorded on a
Shimadzu ESI-MS instrument. UV–Vis absorption spectra were recorded
on a Shimadzu UV-1800 spectrophotometer. Fluorescence spectra were
recorded on an Agilent Cary Eclipse fluorescence spectrophotometer.
The temperature changes of samples were recorded by an infrared
imager (FLIR E6). All measurements were carried out at room temper-
ature in the ambient atmosphere.
2. Experimental section
2.1. Materials and instruments
4-chlorophenylhydrazine hydrochloride, 4-bromophenylhydrazine
hydrochloride, 4-iodophenylhydrazine hydrochloride, and 6-bromohex-
anoic acid were obtained from Heowns-opde Co., Ltd. (Tianjin, China).
3-methyl-2-butanone, cyclohexanone, and sodium acetate were
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H. Liu et al.
Dyes and Pigments 190 (2021) 109327
Fig. 5. The CLSM images of HeLa cells incubated with IR6 (40 μg/mL).
2.2. Preparation of IR6
mixture and stirred for 1 h at room temperature. The yellow solid was
precipitated, then filtered and washed with water. The purified A was
obtained by recrystallization from dichloromethane. (3.69 g, 85.6%). 1H
NMR (400 MHz, DMSO‑d6) δ 2.34 (t, J = 6.2 Hz, 4H), 1.57 (q, J = 6.2 Hz,
2H).
We prepared three halogenated derivatives of cyanine, whose 5-po-
sition of indole ring was substituted by chlorine (IR6), bromine (IR7),
and iodine (IR8), respectively. In the case of IR6, chlorophenylhydrazine
hydrochloride was refluxed with 3-methyl-2-butanone to produce the
indole 6b, which with 6 bromohexanoic acid resulted in the formation of
derivative 6c by N-alkylation reaction. Meanwhile, 2-chloro-1-formyl-3-
hydroxymethylcyclohexene (A) was prepared by cyclohexanone, phos-
phoryl chloride, and dimethylformamide using Vilsmeier–Haack reac-
tion. Finally, the compound 6c and A were used to produce the target
compound IR6 by a condensation reaction. Besides, compound IR7 and
IR8 were prepared according to the reported method [25,26]. The
synthetic route is shown in Fig. 1b.
2.2.2. Synthesis of 5-chloro-2,3,3-trimethyl-3H-indole (6b)
4-chlorophenylhydrazine hydrochloride (6a) (4 g, 22.3 mmol) and 3-
methyl-2-butanone (2.3 mL, 26.7 mmol) were added to a 100 mL
double-necked flask. Afterwards, ethanol (25 ml) and sulfuric acid (3 ml,
95%) was added. The mixture was refluxed at 85 ◦C for 5 h and was
evaporated in vacuum. The brown solution was washed three times with
saturated NaHCO3 solution and water. The organic layer was dried over
Na2SO4 and evaporated. A thick red-brown liquid was the purified
compound 6b (3.59 g, 83.02%). 1H NMR (400 MHz, CDCl3) δ 7.40 (s,
1H), 7.25 (d, J = 2.2 Hz, 1H), 7.22 (s, 1H), 2.24 (s, 3H), 1.28 (s, 6H).
2.2.1. Synthesis of 2-chloro-1-formyl-3-hydroxymethylcyclohexene (A)
In a 250 mL three-necked flask, DMF (24 mL) was added to and
cooled in an ice bath at 0 ◦C. Then, POCl3 (4.6 g, 30 mmol) was added
and the mixture was stirred for 30 min. Cyclohexanone (2.5 g, 25 mmol)
was added and the solution was refluxed at 80 ◦C for 4 h. The red
mixture was slowly poured into a 100 ml beaker with 50 g ice water
2.2.3. Synthesis of 1-(5-carboxypentyl)-5-chloro-2,3,3-trimethyl-3H-indol-
1-ium (6c)
In 100 mL double-necked flask, 5-chloro-2,3,3-trimethyl-3H-indole
(6b) (3.13 g, 16.2 mmol) and 6-bromohexanoic acid (3 mL, 21.7
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H. Liu et al.
Dyes and Pigments 190 (2021) 109327
Fig. 6. Fluorescence microscope images of HeLa cells incubated with IR6, IR7, and IR8 with laser irradiation (right) or without (left). The scale bar is 50
μ
m.
mmol) were dissolved in 30 mL of acetonitrile. The mixture was refluxed
at 85 ◦C for 60 h under shading. Then, the mixture was cooled to room
temperature and evaporated in vacuum. The crude material was purified
by normal phase flash chromatography with CH2Cl2: MeOH (15:1 v/v)
for 6c to obtain a purple liquid (3.67 g, 73.4%). 1H NMR (400 MHz,
DMSO‑d6) δ 8.07 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.72 (dd,
J = 8.6, 2.1 Hz, 1H), 4.45 (t, J = 7.8 Hz, 3H), 2.85 (s, 3H), 2.23 (t, J =
7.2 Hz, 3H), 1.88–1.77 (m, 3H), 1.55 (s, 8H), 1.47–1.37 (m, 3H). 13C
NMR (500 MHz, DMSO‑d6) δ 197.86, 175.07, 144.64, 140.63, 135.00,
129.69, 124.76, 55.13, 48.44, 34.06, 27.55, 26.04, 24.71, 22.50.
Electro-Spray ionization tandem mass spectrometry (ESI-MS) m/z
found: [M+H]+ 308.1414; molecular formula C17H23ClNO+2 requires
[M+H]+ 308.1412.
0.37 mmol) were dissolved in acetic anhydride (16 mL). Then, the
mixture was heated at 70 ◦C and stirred for 2 h under an N2 atmosphere
to obtain the cyanine. Afterwards, the green solution was cooled to room
temperature, and acetic anhydride was removed by rotavap. The
mixture was purified by normal phase flash chromatography with
CH2Cl2: MeOH (20:1 v/v) to obtain the purified IR6 (160 mg, 53.1%). 1H
NMR (400 MHz, MeOD) δ 8.41 (d, J = 14.0 Hz, 2H), 7.58 (d, J = 2.0 Hz,
2H), 7.42 (dd, J = 8.5, 2.0 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 6.29 (d, J =
14.1 Hz, 2H), 4.16 (t, J = 7.4 Hz, 4H), 2.72 (t, J = 6.2 Hz, 4H), 2.28 (t, J
= 7.3 Hz, 4H), 1.98–1.91 (m, 2H), 1.89–1.79 (m, 4H), 1.72 (s, 12H),
1.70–1.65 (m, 4H), 1.48 (dd, J = 10.4, 4.8 Hz, 4H). 13C NMR (400 MHz,
CD3OD) δ 172.58, 150.11, 144.20, 143.08, 141.02, 130.64, 127.36,
122.74, 112.12, 101.37, 49.35, 43.92, 34.12, 26.79, 26.66, 26.04,
24.58. Electro-Spray ionization tandem mass spectrometry (ESI-MS) m/z
found: [M+H]+ 753.2814; molecular formula C42H50Cl3N2O+4 requires
[M+H]+ 753.2801.
2.2.4. Synthesis of 1-(5-carboxypentyl)-2-((E)-2-((E)-3-(2-((E)-1-(5-
carboxypentyl)-5-chloro-3,3dimethylindolin-2-ylidene)ethylidene)-2-
chlorocyclohex-1-en-1-yl)vinyl)-5-chloro-3,3dimethyl-3H-indol-1-ium
(IR6)
2.3. UV–vis and fluorescence spectroscopy
In 100 mL double-necked flask, a mixture of 6c (281.2 mg, 0.36
mmol), A (31.1 mg, 0.2 mmol) and anhydrous sodium acetate (30.4 mg,
IR6, IR7, and IR8 solutions (5 μg/mL) were prepared by dissolving
5

H. Liu et al.
Dyes and Pigments 190 (2021) 109327
1 mL centrifuge tubes and irradiated by a NIR laser (808 nm, 1.0 W/cm2)
for 14 min at room temperature. The laser was then switched off, and the
solutions were cooled naturally. The temperatures of the solutions
during laser irradiation and cooling process were both recorded in
real-time using a FLIR E6 thermal imager.
The photothermal conversion efficiency (
η
) of dyes was evaluated
according to a reported method [28,29]. The
using the following equation:
η of dyes was calculated
ꢀ
)
hA ΔTmax,mix ꢀ ΔTmax,H
2O
ꢀ
)
η
=
I 1 ꢀ 10ꢀ A
808
τs=m
C
D
D
hA
h is the heat transfer coefficient, A is the surface area of the container,
and the value of hA is calculated. ΔTmax,mix and ΔTmax,H Oare the tem-
2
perature changes of the dispersion and solvent at the maximum steady-
state temperature, respectively, I is the laser power and A808 is the
absorbance of dyes at 808 nm mD and CD are the mass and heat capacity
(4.2 kJ/kg) of the deionized water used as the solvent.
Fig. 7. Cell viability of HeLa cells incubated with IR6, IR7, and IR8 for 24 h.
2.6. Photothermal stability
The solutions of IR6, IR7, and IR8 (80 μg/mL) were placed in 1 mL
centrifuge tubes and irradiated by a NIR laser (808 nm, 1.0 W/cm2) for
14 min. The laser was then switched off, and the solution was cooled to
room temperature naturally. This operation was repeated four times.
2.7. Cellular uptake test
To assess the cellular uptake of IR6, IR7, and IR8, HeLa cells (1 × 105
cells/mL) were seeded into 48-well plates and cultured for 24 h. After
that, the fresh medium containing sample concentrations of dyes (IR6,
IR7, and IR8, 40 μg/mL) were substituted for the original culture me-
dium and incubated for different time (0.5 h, 2 h, and 6 h). Then the cells
were washed with PBS for 3 times and treated with a 4% para-
formaldehyde solution (0.5 mL) for 20 min. Subsequently, the cells were
stained with 4,6-diamino-2-phenyl indole (DAPI, 1 μg/mL) solution for
10 min. Finally, the cellular uptake of cells in each well was observed
with the confocal laser scanning microscopy (CLSM).
2.8. Cellular singlet oxygen detection
Fig. 8. Cell viability of HeLa cells incubated with IR6, IR7, and IR8 with laser
The level of intracellular singlet oxygen was detected by 2,7-dichlor-
ofluorescein diacetate (DCFH-DA), which can be oxidized to dichloro-
fluorescein (DCF) in the presence of singlet oxygen [30]. HeLa cells were
firstly cultured for 24 h in a 48-well plate. Then the original medium was
irradiation.
the appropriate amount of each dye in methanol, N,N-dimethylforma-
mide (DMF), and water. Absorption spectra were measured by a UV–Vis
spectrophotometer (UV-1800, Shimadzu, Japan). Fluorescence spectra
were recorded on an Agilent Cary Eclipse fluorescence
spectrophotometer.
replaced with a fresh medium containing IR6, IR7, and IR8 (40 μg/mL)
and incubated for 4 h. After that, HeLa cells were washed with PBS for 3
times and incubated with DCFH-DA for 20 min. Finally, the cells were
irradiated with an 808 nm laser (1.0 W/cm2), and the fluorescence
images of the cells were observed using a fluorescence microscope.
2.4. Singlet oxygen detection in vitro
2.9. Cell culture and cytotoxicity test
To evaluate the generation of singlet oxygen from samples, 1,3-
diphenylisobenzofuran (DPBF, 1 mg/mL) was used as a singlet oxygen
The cell cytotoxicity of dye was evaluated by the methyl thiazo-
lyltetrazolium (MTT) assay [31]. HeLa cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Thermal Fisher Scientific). HeLa cells
catcher. The mixture solutions of DPBF with IR6, IR7, and IR8 (10 μg/
mL) in water and DMF were irradiated using 808 nm laser (0.4 W/cm2).
The degradation of DPBF was determined by a UV–Vis spectrophotom-
eter at different times (0, 1, 2, 3, 4, 5, 7, 10 min).
in 96-well plates (100
μ
L, 1 × 105 cells/mL) were incubated for 24 h
under hypoxic condition (37 ◦C, 5% CO2). Then, IR6, IR7, and IR8 with
different concentrations (0, 10, 20, 40, 80, 100
μg/mL) dissolved in
2.5. Photothermal effect
DMEM were added to the wells and incubated for 24 h. After that, MTT
solution (100 μL, 0.5 mg/mL) was added to each well and the plate was
The photothermal properties of IR6, IR7, and IR8 were determined
using a reported method [27]. Briefly, the solutions of IR6, IR7, and IR8
incubated for an additional 4 h. Each well was then washed with
phosphate-buffered saline (PBS) and added dimethyl sulfoxide (DMSO,
at different concentrations (0, 10, 20, 40, and 80
μ
g/mL) were placed in
100 μL). After the formazan was fully dissolved, the absorbance of each
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H. Liu et al.
Dyes and Pigments 190 (2021) 109327
sample at 490 nm was recorded using a plate reader.
mass of halogen atoms, which further confirmed the previous analysis.
2.10. PTT and PDT synergistic therapy in vitro
3.4. Photothermal property
HeLa cells were seeded in a 96-well plate (1 × 105 cells/mL). After
24 h of incubation, the medium was replaced with fresh medium con-
taining dyes (IR6, IR7, and IR8) with different concentrations (0, 20, 40,
The photothermal effects of IR6, IR7, and IR8 were evaluated. As
shown in Fig. 4a, the temperature of IR6, IR7, and IR8 solutions (40 μg/
mL) increased 27.3 ◦C, 37.1 ◦C, and 42.7 ◦C. This result indicated the
excellent photothermal property of IR6, IR7, and IR8. Therefore, it was
concluded that the photothermal effects showed a positive correlation
with the increased relative atomic mass of halogen atoms. The photo-
80, 100 μg/mL) and incubated for 12 h. Then, each well was irradiated
using a NIR laser (808 nm, 1.0 W/cm2) for 5 min. After that, the cells
were washed with PBS. Finally, the MTT solution (100 μL, 0.5 mg/mL)
was added to each well and cultured for 4 h to evaluate the photo-
thermal and photodynamic effects in vitro.
thermal conversion efficiency (η) of IR6, IR7, and IR8 solutions was
calculated as 42.3%, 43.4%, 46.6%, which further confirmed the
experiment results.
3. Results and discussion
Then we evaluated the effect of IR6, IR7, and IR8 concentration on
temperature rise (Figs. S16b, c, and d). IR6, IR7, and IR8 all displayed
different degrees of temperature rise with increasing concentration.
In order to investigate the photothermal stability of dyes, IR6, IR7,
and IR8 were irradiated by the multiple laser with recording the irra-
diated temperature’ changes. However, the maximum temperatures of
IR6, IR7, and IR8 showed varying decreased degrees during four cycles.
The differences between the first and last cycles of the maximum tem-
perature of IR6, IR7, and IR8 were 17.3 ◦C, 9.5 ◦C, and 8.7 ◦C, respec-
tively. The experiment results indicated that IR6, IR7, and IR8 had not a
good photostability, which were positively related to the increased
relative atomic mass of halogen atoms.
3.1. Synthesis and characterization
Halogenated cyanine dyes (IR6, IR7, and IR8) were synthesized by
introducing Cl, Br, or I atoms into position 5 of the indole skeleton,
respectively. The general synthesis procedures for IR6, IR7, and IR8 are
shown in Fig. S1. Besides, compound IR7 and IR8 were prepared ac-
cording to the reported method [25,26]. The structures of all dyes were
confirmed by 1H NMR, 13C NMR, ESI-MS, and MALDI-TOF-MS analysis.
3.2. Optical properties
3.5. Cellular uptake
The absorption and fluorescence spectra of IR6, IR7, and IR8 were
obtained. As shown in Fig. 2a and b, the maximal absorption peaks of
IR6, IR7, and IR8 were located at 790, 793, and 796 nm in methanol.
However, the emission peaks of IR6, IR7, and IR8 were at 836, 837, and
844 nm, respectively. We can observe that the introduction of heavy
atoms will make the absorption and emission wavelength of dyes
redshift, which was positively correlated with the increase of the atomic
number of the halogen atom.
The uptakes of HeLa cells to IR6, IR7, and IR8 were investigated
using the confocal laser scanning microscopy (CLSM). As shown in
Fig. 5, after having incubated with IR6 for 0.5 h, 2 h, and 6 h, the
fluorescence brightness (FB) obviously increased in the red fluorescence
channel. This means cellular uptake of IR6 gradually increased over
time. Similarly, as shown in Figs. S17 and S18, HeLa cells incubated with
IR7, and IR8 also showed increased cellular uptake to dyes over time.
3.3. Singlet oxygen generation in vitro
3.6. Cellular singlet oxygen detection
To evaluate the photodynamic properties of IR6, IR7, and IR8, DPBF
was used to detect the singlet oxygen generation of dyes. As a singlet
oxygen detector, DPBF can occur degradation when it reacted with
singlet oxygen. Therefore, the change in the absorbance of DPBF can be
used to observe the generation of singlet oxygen. The change in absor-
bance of DPBF under laser irradiation in water and DMF was negligible,
indicating that no singlet oxygen was produced (Fig. S14a). The absor-
bance of DPBF with IR6, IR7, and IR8 displayed a significant drop at 418
nm under irradiation of NIR light, confirming their ability to generate
singlet oxygen (Figs. S14b, c, and d). The singlet oxygen generation of
IR8 was higher than that of IR6 and IR7 (The absorbance of DPBF was
reduced by 78.3%, 63.4%, and 32.6%, respectively), indicating that the
iodine atom had the greatest impact on generating singlet oxygen. Be-
sides, it was worth noting that IR6 has a better singlet oxygen generation
ability than IR7. The reason may be that cyanine dye with bromine atom
was more likely to form J-aggregates in aqueous systems, resulting in a
decrease in the lifetime of the triplet excited state, decreasing ability for
singlet oxygen generation [32–34].
The generation of singlet oxygen in cells under the laser irradiation
was detected using DCFH-DA (2,7-dichloro-dihydro-fluorescein diac-
etate). DCFH-DA would be hydrolyzed into non-fluorescent DCFH (2,7-
dichlorofluorescin) in the cell, which was oxidized by singlet oxygen
into green fluorescent DCF (dichlorofluorescein). Fig. 6 showed the cells
incubated with IR6, IR7, and IR8 possessed negligible green fluores-
cence without laser irradiation. In contrast, the cells with laser irradia-
tion showed bright green fluorescence. These results showed that IR6,
IR7, and IR8 could produce singlet oxygen effectively in cells and had
great potential for PDT.
3.7. Cell cytotoxicity in vitro
MTT assay was used to reveal the cell cytotoxicity of IR6, IR7, and
IR8. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-htetrazolium
bromide) would be reduced to formazan (purple solid could dissolve in
DMSO) by succinate dehydrogenase in living cells [31]. As shown in
Fig. 7, different concentrations of IR6, IR7, and IR8 were added to HeLa
cells, MTT value was measured after cell incubation for 24 h. Even with
Therefore, we measured the absorption spectra of IR6, IR7, and IR8
in aqueous solutions. As shown in Fig. 2c, IR6 only had its monomer
absorption peak at 788 nm. However, IR7 not only had the monomer
absorption peak in 800 nm but also showed the absorption peak of the J-
aggregates at 926 nm. This proved that IR7 formed J-aggregates in the
aqueous solution, causing the aggregation-caused quenching (ACQ) ef-
fect and reducing the generation of singlet oxygen. As shown in Fig. 2d,
we only found that the monomer absorption peaks of IR6, IR7, and IR8
were located at 800, 802, and 806 nm in DMF. Meanwhile, as shown in
Fig. 3b, it was observed that the singlet oxygen generation of IR6, IR7,
and IR8 showed a positive correlation with the increased relative atomic
concentration at 100 μg/mL, the cell viability of IR6, IR7, and IR8 were
still over 80%, indicating the low cytotoxicity of IR6, IR7, and IR8.
3.8. PTT and PDT synergistic therapy in vitro
The phototherapy effects of IR6, IR7, and IR8 were investigated
against HeLa cells using standard MTT assay. As shown in Fig. 8,
different concentrations of IR6, IR7, and IR8 were added to HeLa cells,
and the cells were irradiated with NIR irradiation for 5 min. The MTT
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value was measured after 24 h. IR8 showed a more effective cytostatic
effect (half-maximal inhibitory concentration, IC50 = 16.2 μg/mL) than
IR6 and IR7 in agreement with the photothermal and photodynamic
experiment results in solution-based. However, IR6 displayed better
phototherapy effects than IR7, possibly due to IR7 can effectively
generate higher singlet oxygen.
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Through comparison of IR6, IR7, and IR8 in solution-based and cell-
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4. Conclusion
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In summary, we synthesized three halogenated cyanine dyes through
general procedures and evaluated the influence of heavy atoms on the
photothermal and photodynamic performance of cyanine. The intro-
duction of heavy atoms would cause a redshift in the absorption and
emission wavelength of the cyanine. Meanwhile, the effect of heavy
atoms on the singlet oxygen generation capacity, photothermal con-
version efficiency, and photothermal stability was positively correlated
with the increase of the atomic number of the halogen atom. IR6, IR7,
and IR8 all exhibited low dark toxicity. Finally, cyanine IR8 undoubt-
edly exhibited the best reactive oxygen generation capacity, photo-
thermal conversion rate, and therapeutic efficiency over IR6, and IR7.
The influence of heavy atoms on cyanine dyes of this work can provide
guidance to design other phototherapy agents with high photodynamic
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgements
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This work was financially supported by the National Natural Science
Foundation of China (NSFC, Nos. 21676187).
Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109327.
Author statement
[20] Lin W, Li Y, Zhang W, Liu S, Xie Z, Jing X. Near-infrared polymeric nanoparticles
with high content of cyanine for bimodal imaging and photothermal therapy. ACS
Appl Mater Interfaces 2016;8(37):24426–32. https://doi.org/10.1021/
acsami.6b07103.
Hao Liu: Conceptualization, Methodology, Software, Validation,
Investigation, Formal analysis, Resources, Data curation, Writing –
original draft, Writing – review & editing. Juanjuan Yin: Software, Re-
sources. Enyun Xing: Validation. Yingying Du: Validation, Formal
analysis. Yu Su: Validation. Yaqing Feng: Conceptualization, Writing –
review & editingWriting Review and Editing, Visualization, Supervision,
Project administration, Funding acquisition. Shuxian Meng: Conceptu-
alization, Methodology, Writing – review & editingWriting Review and
Editing, Visualization, Supervision, Project administration, Funding
acquisition
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