A near-infrared heptamethine aminocyanine dye with a long-lived excited triplet state for photodynamic therapy

Article abstract of DOI:10.1039/c8cc04582h

A water-soluble near-infrared aminocyanine dye has been developed with a long triplet-state lifetime (τ = 9.16 μs in deaerated ethanol). Thereby, extremely high singlet oxygen quantum yield (Φ_Δ = 0.20) and low dark cytotoxicity (IC₅₀ = 715.4 μM) were achieved. The potential of the dye as a PDT photosensitizer was demonstrated.

Full text of DOI:10.1039/c8cc04582h

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DOI: 10.1039/C8CC04582H
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A near infrared heptamethine aminocyanine dye with a long-
living excited triplet state for photodynamic therapy
Long Jiao, Fengling Song*, Jingnan Cui, Xiaojun Peng
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
water-soluble near-infrared aminocyanine dye have been also used as photosensitizers for PDT, like ICG (approved by the
developed with a long triplet-state lifetime (τ = 9.16 μs in FDA in the United States for medical diagnostic application).
deaerated ethanol). Thereby, extremely high singlet oxygen However, they have low phototoxicity because they are
quantum yield (Φ△ = 0.20) and low dark cytotoxicity (IC50 = 715.4 characterized by extremely low singlet oxygen quantum yield,
μM) were achieved. The potential of the dye as
photosensitizer was demonstrated.
a
PDT which has limited their practical utility for PDT.13 Heavy atoms
are introduced in the structural skeleton of Cy7 dyes, which is
known to efficiently increase ISC process and significantly
improve singlet oxygen generation.14-16 But the resulting
modified dyes usually have high dark cytotoxicity. 2,2,6,6-
Tetramethylpiperidinyloxy (TEMPO) is a stable radical which is
widely used as a nitroxide spin label in obtaining biochemical
reaction information in vivo due to its low cytotoxicity.17,18
Meanwhile, TEMPO is known to enhance inter system crossing
(ISC) process for triplet-state photosensitizers.19-22 So far as we
know, introducing TEMPO into Cy7 dye to greatly enhance ISC
efficiency for PDT purpose has not been reported yet.
Photodynamic therapy (PDT) has become an effective
malignant cancers treatment approach with minimal invasion
and high spatiotemporal precision.1-3 PDT calls for excellent
photosensitizers with low dark cytotoxicity and good
phototoxicity.4 Nevertheless, the traditional photosensitizer
molecules are mostly hydrophobic molecules with high dark
cytotoxicity, which greatly restricts their clinical application and
promotion.5 For example, Photofrin,
a porphyrin-based
photosensitizer, has complex chemical nature and
nonnegligible intracellular accumulation, especially retentions
by skin which leads to protracted cutaneous photosensitivity.
Therefore, patients were usually required to avoid light
exposure for a long time after PDT. Otherwise, it will cause
skin allergy and eye damage.6,7 Besides the toxicity, the
photosensitizers are expected to be irradiated by near-infrared
(NIR) light. Because working in NIR optical window can provide
deeper irradiation depth. In this context, as an ideal and
effective photosensitizer for PDT, low dark toxicity but
prominent phototoxicity, and strong absorption in NIR region
(600-900 nm) are extremely important prerequisites.
Herein we designed and synthesized a heptamethine
aminocyanine dye
hydrophilic known Cy7 dye
2
containing the TEMPO substituent from a
according to our previous reported
1
method (Scheme 1).23,24 We aim to provide an efficient NIR PDT
photosensitizer with high singlet oxygen quantum yield as well
as low dark toxicity for application in oncology.
Heptamethine cyanine dyes (Cy7) such as indocyanine green
(ICG), IR-780, IR-783, IR-808, IR-820, IR-825 have emerged as an
excellent NIR imaging agents in fluorescence diagnosis
applications due to their low cytotoxicity.8-12 Some of them are
Scheme. 1 Synthesis of heptamethine aminocyanine dye 2.
After the preparation of dye
1 and dye 2 (Scheme S1), their
photophysical properties were tested. As we expected, dye
possesses a large Stokes shift (~100nm) due to the introduction
of 4-amino-TEMPO at the center of the conjugated bridge of dye
2
a.Address: State Key Laboratory of Fine Chemicals Dalian University of Technology
No. 2 Linggong Road, High-tech District, Dalian,116024, China.
b.E-mail: songfl@dlut.edu.cn;
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: Synthesis and
characterization of compounds. HPLC data, Transient absorption measurements,
Hela cells culture and staining, Cofocal fluorescence imaging. Cytotoxicity assays.
See DOI: 10.1039/x0xx00000x
1
, Meanwhile, dye 1 has a small Stokes shift (~30nm) (Figure
S4). This result of enlarging Stokes shift is consistent with our
previous work about heptamethine aminocyanine dyes.23,24
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Still, the absorption and fluorescence peaks of the dye
Journal Name
2
are in singlet oxygen, the singlet oxygen generation abilVitieiwesAroticfledOynelin1e
were measured tDhOroI:u1g0h.1039/uCs8inCgC04518,23H-
NIR range, which makes it suitable for bio-applications, and dye
2
including PDT for tumors.8,25,26
To affirm the PDT potential of dye
diphenylisobenzofuran (DPBF) as singlet oxygen trapping agent.
2
, we try to arrest its triplet The absorbance of DPBF at 410 nm decreased remarkably in the
excited state by nanosecond time-resolved transient difference presence of dye
2 under irradiation (Figure S6a), while the
absorption spectra. Because a good PDT photosensitizer absorbance decline of DPBF at 410 nm is extremely slow in the
1
normally has a long-living triplet excited state for sensitizing the presence of dye
molecule oxygen in tumor region, which would generate generation efficiency of both dye
cytotoxic reactive oxygen species (ROS, such as singlet oxygen) linear relationship. But, the singlet oxygen generation ability of
to induce cancer cells death. As shown in Figure 1, a salient dye is extremely prominent when compared to dye
1
(Figure S6b). As seen from Figure 2, the O2
1
and dye 2 shows a nearly
2
1.
feature of transient absorption was found between 630 nm and Meanwhile, 4-amino-TEMPO as a control does not cause
820 nm after 532 nm laser excitation. Two bleaching peaks are obvious bleaching of DPBF under LED irradiation for 700 s.
located at ~660 nm and ~790 nm, respectively. The former These results support our expectation that introducing the
absorption centred at 660 nm can be assigned to the bleaching radical of nitroxide into heptamethine cyanine dye can enhance
of ground state. The later one at 790 nm should be attributed the ISC process of cyanine dyes to produce more 1O2. To
to the transient absorption of the formed long-living triplet quantify the singlet oxygen generation abilities of dye
excited state ( = 9.16 μs under argon, blue kinetic fitting curve , the singlet oxygen quantum yield (Φ△) of dye and dye
in Figure 1b). And this long-living transient was found to be were calculated by choosing methylene blue in ethanol with
sensitive to oxygen ( has a considerably higher
= 0.38 μs under air, green kinetic fitting ΦMB = 0.52 as reference.32,33 Dye
curve in Figure 1b), which also confirms that this microsecond singlet oxygen quantum yield (Φ△ = 0.20) than dye (Φ△
1 and dye

2
1
2

2
1
=
scale transient is the formed triplet excited state specie. Under 0.006) in ethanol. So far as we know, the singlet oxygen
the same experimental conditional of nanosecond time- quantum yields (Φ△) of other cyanine dyes like ICG and IR-783
resolved transient difference absorption spectra, no salient used for PDT are only 0.008 and 0.007, respectively.26
signal was observed for dye
that efficient radical-enhanced intersystem crossing occur in
dye due to the radical substituent, thereby obtaining a long-
1 (Figure S5). These results indicate
2
living excited triplet state. Such a long-living triplet excited state
has not been achieved by the conventional methods relying on
heavy atom effect to enhance ISC process of cyanine dyes.14-16,
27,28
Enhanced ISC by the introduced TEMPO group for dye
2
should be attributed to electron spin polarization (ESP) based
on a radical-triplet pair mechanism (RTPM).29.30,31 As far as we
know, such a microsecond scale transient absorption has never
been observed in reported cyanine dyes.
Fig. 2 The bleaching rate comparison of DPBF at 410 nm. A NIR
LED array (

ex = 660 nm; 0.8 mW/cm2) for different irradiation
and dye were respectively dissolved in absolute
times. Dye
1
2
ethanol to a final concentration with almost the same
absorbance (~0.1) at 660 nm and mixed with DPBF (50 μM). 4-
Amino-TEMPO (3 μM) was used with the same concentration as
dye 2. Data are presented as the mean value ± SD (n = 3).
To be used in bio-applications, the chemical durability of dye
2
should be evaluated. Because the meso position substitution
of the Cy7 fluorophores has been doubted to be labile when
such a Cy7 dye is circulating throughout the blood stream.
Fig. 1 (a) Nanosecond time-resolved transient difference
absorption spectra of dye
nm laser pulse, decay times as indicated; (b) Life decay curves
of transient species in dye (10.0 μM) under argon (blue line)
2 (10.0 μM in deaerated ethanol). 532
Besides, reducing substances in the blood may cause dye
be reduced into dye 2-OH (Scheme S1). We found the reduction
product dye 2-OH shows better ionization capacity than dye
2 to
2
2
and air atmosphere (green line). Blue line shows the profile
after deoxygenating with argon bubbles (τ = 9.16 μs, residuals =
0.75) and green line shows the profile after the oxygen-free
sample was settled for 24 h under air atmosphere (τ = 0.38 μs,
under high resolution mass spectra (HRMS) condition (Figure
S1, S2). Therefore, HRMS was employed to evaluate the
chemical stability of dye 2. Dye 2 was treated with 100% ICR
residuals = 0.82). Excited at 532 nm and monitored at 800 nm. mice serum for 4 h and 24 h before evaluation. For comparison,
All measurements are performed at room temperature.
a control sample by mixing dye 2-OH and dye
2 (mole ratio of
dye 2-OH : dye is 1:9) was also checked by HRMS. As shown in
Figure S7, the results of HRMS data indicate that the content of
possible reduction product was far below 10% even after 24 h
2
We know that production of reactive oxygen species (ROS) is
strongly linked to the excited triplet states of the
photosensitizer. Therefore, to confirm the ability of dye
2 with
treatment with serum. It means that dye
2 has a robust
a long-living excited triplet state may sensitize more oxygen into
2 | J. Name., 2012, 00, 1-3
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chemical stability and can be used for PDT applications in using Dihydroethidium (DHE) staining. DHE is a ROS fluorescent
View Article Online
biological environment.
probe. After DHE being oxidized by RDOOSI,: 1t0h.1e03c9e/Clls8’CCn0u4c5le82uHs
Based on excellent chemical durability of dye
2
, we should exhibit bright fluorescent signal. As shown in Figure S10,
subsequently try to verify dye 2 a better photosensitizer for PDT the oxidized DHE migrated from the cytoplasm (green arrow) to
in living cells. It is desirable to develop photosensitizers with low the nucleus (red arrow) and produced a bright red fluorescence
dark cytotoxicity. The amphiphilic dye molecules ICG with low during the process of PDT. Meanwhile, HeLa cell morphology
dark cytotoxicity as a photosensitizer for PDT has been changed significantly after light exposure, accompanying by
studied.26-28,32-34 However, its low singlet oxygen quantum yield nuclear condensation. These results indicate that dye
(Φ△ = 0.008) limits its practical application. Protoporphyrin IX enter the cells and produce ROS during irradiation, although it
(PpIX), known PDT photosensitizer, has an excellent has water solubility.
photocytotoxicity. But its high dark cytotoxicity is the mainly
side effect for PDT. Here we evaluated both phototoxicity and
dark cytotoxicity of dye , dye and PpIX towards HeLa cells by
MTT assays. As shown in Figure 3a, dye did not show
significant photocytotoxicity to HeLa cells with IC50 value at
2 can
a
,
1
2
1
429.6 μM. The dose-dependent phototoxicity of dye
recorded with IC50 value at 14.2 μM, which tells that dye
2
2
was
has
a moderate phototoxicity and can induce a time-dependent
process of cell death (Figure S8). Although PpIX showed a much
higher phototoxicity with IC50 value at 5.9 μM than dye
the dark cytotoxicity of PpIX is much higher than that of dye
dye (Figure 3b, IC50 of dye and were 659.7 and 715.4 μM
2. But,
1
,
2
1
2
respectively, while that of PpIX was 76.3 μM). The dark
cytotoxicity of dye 2 was similar with that of dye 1 after the
introduction of 4-amino-TEMPO. The phototoxicity index (PI)
(the ratio of IC50 of dark cytotoxicity to IC50 of phototoxicity) are
Fig. 4 Morphology of HeLa cells stained by acridine
orange/ethidium bromide (AO/EB) after PDT treatment under
confocal fluorescence imaging. Dye 2 (20 μM) was co-cultivated
with Hela cells for 12 h, then HeLa cells were irradiated for 20
min before stained with AO/EB. LED array irradiation (λex = 660
nm, power density of 50 mW/cm2). Control A-HeLa cells were
calculated for PpIX (PI = 12.9), dye
1 (PI = 1.5) and dye 2 (PI =
50.4) to evaluate their potentials as PDT photosensitizers.
Although PpIX have a high phototoxicity, a high dark toxicity
makes its PI value lower than that of dye
2. This result suggests
that dye should be a better photosensitizer for clinical PDT.
2
irradiated for 20 min without dye
HeLa cells were co-cultivated with dye
2
co-cultivation; Control B-
for 12 h without
2
irradiation; Group A-HeLa cells were immediately stained by
AO/EB after irradiation; Group B-HeLa cells were stained by
AO/EB after continuing incubation for 6 hours after irradiation.
VN-viable cells; VA-early apoptotic cells; NVA-late apoptotic
cells; NVN-necrotic cells. Scale bar = 20 μm.
At last, we tried to demonstrate the practical feasibility of
dye
2 as a PDT photosensitizer in a visual mode by using
Fig. 3 (a) Phototoxicity evaluation of dye 1, dye 2 and PpIX on HeLa
cells for 20 minutes irradiation (ex = 660 nm, 50 mW/cm2), MTT array
was measured after another 6 h incubation. (b) Dark cytotoxicity
evaluation of dye 1, dye 2 and PpIX on HeLa cells. Data are presented
as the mean value ± SD (n = 6).
fluorescence confocal imaging experiments. Acridine orange
(AO) stains the DNA and RNA of living cells and gives out green
fluorescence. Ethidium bromide (EB) stains DNA and RNA of
dead cells and gives out red fluorescence. Through the cells’
AO/EB staining, the quantization differences of living cells (VN),
necrotic cells (NVN), early apoptotic cells (VA) and the late stage
apoptotic cells (NVA) can be visually distinguished by confocal
fluorescence microscopy (Figure 4). As shown in Figure 4
(control A), HeLa cells appeared as irregular shuttle with
uniformly green fluorescence of AO after light irradiation, while
the characteristic red fluorescence of EB was not detected.
These results indicate light irradiation from the LED light source
used in our experiments cannot cause phototoxicity. In the
After high phototoxicity and low dark toxicity has been
confirmed, dye 2 was investigated to clarify the mode of action
for its PDT effect. According to our design, the low dark toxicity
should be due to its good water solubility. It is curious to know
whether the dye
phototoxicity is achieved. HeLa cells were chosen again to be
co-stained with dye and DAPI, a nuclear fluorescence dye. As
shown in the confocal fluorescence images of Figure S9, the dye
was internalized and uniformly stained in the region of
2 can enter the living cells, and how its high
2
samples of control B, HeLa cells were stained with dye
for 12 h without light irradiation. No obvious cytotoxicity was
observed either, which supports that dye at the used
2 (20 μM)
2
cytoplasm. Then, we carried out the PDT to check whether
intracellular reactive oxygen species (ROS) was generated by
2
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Journal Name
Josephson, B. Knudsen, M. Tighiouart, H. L. Kim, H. E. Zhau, L. W.
concentration do not induce cells death. Meanwhile, after PDT
under the same light irradiation and at the same concentration
of dye 2, early apoptotic cells (VA) are dominated right after
irradiation (Group A), and obvious increased late stage
apoptotic cells (NVA) and some necrotic cells (NVN) can be
observed for cells with another 6 h incubation after irradiation
(Group B). Taken together, these results suggest that cells death
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2
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In summary, we have developed
a near infrared
aminocyanine dye with a long-living excited triplet state (τ =
2
9.16 μs) by introduction of a radical into water-soluble cyanine
dye structure. Its potential in PDT as a photosensitizer has been
evaluated. In the one hand, dye
singlet oxygen quantum yield (Φ△ = 0.20) and a superior PDT
effect in the living cells level. In the other hand, dye has a very
low dark cytotoxicity and a robust chemical stability. After PDT,
dye can induce cell death predominantly via apoptosis. To
fulfil its potential in PDT, an improvement with target function
based on dye is being done. We firmly believe that new
compounds based on dye have a bright prospect for
2 exhibits significantly high
2
2
2
2
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biomedical and clinical applications in the future.
This work was supported financially by the NNSF of China
(21576038, 21421005), the Fundamental Research Funds for
the Central Universities of China (DUT16TD21), and Science
Program of Dalian City (2014J11JH133, 2015J12JH207).
Conflicts of interest
There are no conflicts to declare.
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