Charge transfer NIR dyes for improved photoacoustic effect

Article abstract of DOI:10.1016/j.dyepig.2015.11.003

A new class of NIR absorbing dyes accessed by a cycloaddition-cycloreversion reaction of alkynes with 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane is reported. The photoacoustic effect of the new NIR dyes was systematically studied. The results va

Full text of DOI:10.1016/j.dyepig.2015.11.003

Accepted Manuscript
Charge Transfer NIR Dyes for Improved Photoacoustic Effect
An-Ping Xu, Hui-Hui Han, Jing Lu, Pei-Pei Yang, Yu-Juan Gao, Hong-Wei An, Di
Zhang, Li-Zhong Li, Jing-Ping Zhang, Dong Wang, Lei Wang, Hao Wang
PII:
S0143-7208(15)00430-1
10.1016/j.dyepig.2015.11.003
DYPI 4994
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 19 September 2015
Revised Date: 2 November 2015
Accepted Date: 4 November 2015
Please cite this article as: Xu A-P, Han H-H, Lu J, Yang P-P, Gao Y-J, An H-W, Zhang D, Li L-Z, Zhang
J-P, Wang D, Wang L, Wang H, Charge Transfer NIR Dyes for Improved Photoacoustic Effect, Dyes
and Pigments (2015), doi: 10.1016/j.dyepig.2015.11.003.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A new class of NIR absorbing dyes accessed by a cycloaddition-cycloreversion reaction of alkynes with tetrafluoro-TCNQ is
reported. Studies on the photoacoustic effect of the series of NIR dyes revealed a molar extinction coefficiency which
Charge Transfer NIR Dyes for Improved
Photoacoustic Effect
An-Ping Xu,‡a,c Hui-Hui Han,‡b Jing Lu,d Pei-Pei Yang,a Yu-Juan Gao,a Hong-Wei An,a Di Zhang,a Li-Zhong
b
a
a
Li,c Jing-Ping Zhang,d Dong Wang, Lei Wang, and Hao Wang
aCAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST)
bBeijing Key Laboratory of Function Materials for Molecule & Structure Construction, Department of Material Physics and Chemistry, School of
Materials Science and Engineering, University of Science and Technology Beijing
cKey Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission & Ministry of Education, South-Central University for
Nationalities
dFaculty of Chemistry, Northeast Normal University Changchun,P. R. China
contributed to the displayed photoacoustic effect.

ACCEPTED MANUSCRIPT
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Charge Transfer NIR Dyes for Improved Photoacoustic Effect
An-Ping Xu,‡a,c Hui-Hui Han,‡b Jing Lu,d Pei-Pei Yang,a Yu-Juan Gao,a Hong-Wei An,a Di Zhang,a Li-
b
a
a
Zhong Li,c Jing-Ping Zhang,d Dong Wang, Lei Wang, and Hao Wang
aCAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST)
bBeijing Key Laboratory of Function Materials for Molecule & Structure Construction, Department of Material Physics and Chemistry, School of Materials
Science and Engineering, University of Science and Technology Beijing
cKey Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission & Ministry of Education, South-Central University for Nationalities
dFaculty of Chemistry, Northeast Normal University Changchun,P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
A new class of NIR absorbing dyes accessed by a cycloaddition-cycloreversion
reaction of alkynes with 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane is
reported. The photoacoustic effect of the new NIR dyes was systematically studied.
The results validated the positive correlation between the molar extinction coefficient
and the photoacoustic effect. All of the new NIR dyes showed higher photoacoustic
intensities compared to indocyanine green dye, which is the standard organic dye
widely used as a photoacoustic contrast agent. Finally, the nano-sized liposomes
loaded with the NIR dye showed good stabilities and high photoacoustic intensities,
and were utilized to image the cells in agarose gels for a long time. The study guides
the future design of NIR dyes for photoacoustic molecular materials with high
intensities and displays promising applications in biomedical photoacoustic imaging.
Received in revised form
Accepted
Available online
Keywords:
Near-infrared
Dye
Photoacoustic effect
Bioimaging
Charge transfer
2009 Elsevier Ltd. All rights reserved
.
‡ These authors contributed equally to this work.
* Corresponding authors. Tel./fax: +86 82545759. E-mail addresses: wanghao@nanoctr.cn, (H. Wang), wanglei@nanoctr.cn (L. Wang) wangdong@ustb.edu.cn
(D. Wang).

Introduction
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The photoacoustic (PA) effect was discovered by Alexander Graham Bell in 1880.[1,2] The PA effect refers to the phenomenon
wherein matter absorbs a short-pulsed laser beam and converts its energy into sound (via heat, which induces localized pressure
[3]
changes).
Based on the PA effect, photoacoustic imaging (PAI) has been developed as a highly promising tool for studying
intracellular physiological processes. PAI combines the high spatial resolution of ultrasonic imaging and the high contrast of optical
imaging.[4,5] Both the set-up of PAI devices and PA contrast agents contribute to the imaging performance. Organic dyes with a
maximum absorption in the NIR range, such as cyanine, [6] porphyrin[7] and borondipyrromethene derivatives, [8] are widely used PA
contrast agents. The development of new PA molecular materials are limited because the influence of factors on the PA effect have
seldom been systematically studied, resulting in a lack of guidance for the design of molecular PA contrast agents. [9-11] Recently, we
focused on [2+2] cycloaddition-cycloreversion chemistry, between either tetracyanoethene (TCNE), 7,7,8,8-tetracyanoquinodimethane
(TCNQ) or 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ) and 'electronically confused' alkynes. [12-14] The resulting
adducts, especially those formed from F4-TCNQ, had good solubility and were easily prepared in high yields. Notably, these adducts
showed a strong absorption in the 700-900 nm region, which made them highly suitable contrast agents for PAI. Here we report a new
series of NIR dyes (abbreviated as FTQ) based on F4-TCNQ. The series of FTQ dyes was synthesized by a cycloaddition-
cycloreversion reaction between F4-TCNQ and N,N-dibutyl-4-ethynylanilines (having different substitutions for fine modulation of
optical properties). The PA effect of the series of FTQ dyes was characterized and they exhibited a higher PA intensity than that of
indocyanine green (ICG) under the same experimental conditions. FTQ-4 was successfully utilized for long-term cell imaging when
self-assembled into hydrophobic phospholipid bilayers of liposomes. To further understand PA molecular materials, the relationship
between molecular structures and PA intensities was investigated. The results indicate that substitution with other electron-donating or
electron-withdrawing groups can enhance the molar extinction coefficient and PA intensity, which is in agreement with photothermal
theory. This work provides high performance PA molecular materials and can direct the design of new NIR absorbing dyes for PAI.
2. Experimental
2.1. Materials and methods
Reagents were purchased from commercial sources (Aldrich) and used without further purification. 1H NMR spectra were measured
on a Bruker AV500 NMR spectrometer (500 MHz) at 20 °C. Chemical shifts are reported in ppm downfield from SiMe4, using the
solvent’s residual signal as an internal reference. FT-IR was recorded on a PerkinElmer LR-64912C Fourier transform infrared
spectrophotometer. All MALDI-TOF-MS spectra were measured on a Shimadzu AXIMA-CFR mass spectrometer. The operation was
performed at an accelerating potential of 20 kV by a linear positive ion mode with dithranol as a matrix. UV/Vis/NIR spectra were
recorded in a quartz cuvette on a JASCO V-570 spectrophotometer. Elemental analyses were measured on a FLASH EA 1112
elemental analyzer. The reacting aromatic alkyne molecules are shown in Scheme 1. a1-a4, b4 and FTQ-4 were synthesized according
to literature methods.13,14 The other photoacoustic effect molecules were synthesized and fully characterized by NMR, FT-IR, MS
spectra and elemental analysis from which the chemical structures were verified.

2.2. Synthesis
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2.2.1. Synthesis of N,N-dibutyl-4-(phenylethynyl)aniline (b1). Monomer a4 (137 mg, 0.600 mmol) and bromobenzene (0.079 g, 0.500
mmol) were dissolved in TEA (150 mL) and THF (150 mL). After the solution was flushed with bubbling Ar gas for 30 min,
Pd(PPh3)2Cl2 (12.6 mg, 0.018 mmol) and CuI (5.7 mg, 0.030 mmol) were added. The reaction mixture was then stirred at 80 °C for 10 h
under Ar atmosphere. The resulting mixture was concentrated, diluted with CH2Cl2, and filtered through a plug of silica gel. The solvent
was removed under vacuum and the product was purified by column chromatography (SiO2, petroleum ether/CH2Cl2=4:1) to give b1
(0.115 g, 63%) as a yellow oil liquid.1H NMR (CDCl3, 500 MHz): δ = 1.00 (t, J=7.5 Hz, 6H), 1.43 (m, 4H), 1.63 (m, 4H), 3.33 (t, J=7.5
Hz, 4H), 6.61 (d, J=5.4 Hz, 2H), 7.31 (t, J=7.0 Hz, 1H), 7.36(t, J=7.0 Hz, 2H), 7.40 (d, J=5.1 Hz, 2H), 7.53 (d, J=7.5 Hz, 2H) ppm. FT-
IR (KBr): υ=2947, 2850, 2200, 1587, 1515, 1379, 1160, 926, 804, 523 cm-1. MALDI-TOF-MS (dithranol): m/z: calc’d for C22H27N:
305.21 g/mol, found: 306.3 g/mol [MH]+. Elemental analysis calc’d (%) for C22H27N (305.21): C 86.51, H 8.91, N 4.58; found: C 86.50,
H 8.92, N 4.59.
2.2.2.
Synthesis of 2-(2-(4-(Dibutylamino)phenyl)-2-(4-(dicyanomethylene)-2,3,5,6-tetrafluorocyclohexa-2,5-dien-1-ylidene)-1-
phenylethylidene)malononitrile (FTQ-1). The monomer b5 (0.100 g, 0.328 mmol) and F4-TCNQ (0.090 mg, 0.330 mmol) were
dissolved in chlorobenzene (2 mL). The reaction mixture was then stirred at 100 °C for 2 h under Ar atmosphere. The solvent was
removed under vacuum and the product was purified by column chromatography (SiO2, CH2Cl2) to give FTQ-1 (0.183 g, 96%) as a
1
black red solid. H NMR (CDCl3, 500 MHz): δ = 1.06 (t, J=7.5 Hz, 6H), 1.50 (m, 4H), 1.78 (m, 4H), 3.62 (t, J=6.0 Hz, 4H), 6.87 (d,
J=9.5 Hz, 2H), 7.39 (d, J=9.0 Hz, 2H), 7.54 (t, J=7.5 Hz, 2H), 7.63 (t, J=8.0 Hz, 3H) ppm. FT-IR (KBr): υ=2946, 2850, 2200, 1587,
1515, 1379, 1160, 922, 804, 529 cm-1. MALDI-TOF-MS (dithranol): m/z: calc’d for C34H27F4N5: 581.22 g/mol, found: 582.3 g/mol
[MH]+. Elemental analysis calc’d (%) for C34H27F4N5 (581.22): C 70.21, H 4.68, N 12.04; found: C 70.20, H 4.68, N 12.05.
2.2.3. Synthesis of 4,4'-(Ethyne-1,2-diyl)bis(N,N-dibutylaniline) (b2). a2 (0.166 g, 0.500 mmol) and a4 (0.137 g, 0.600 mmol) were
dissolved in TEA (150 mL) and THF (150 mL). The Pd(PPh3)2Cl2 (12.6 mg, 0.0180 mmol) and CuI (5.7 mg, 0.030 mmol) were added
into the solution after bubbling with Ar gas for 30 min. The reaction mixture was then stirred at 40 °C for 10 h under an Ar atmosphere.
The resulting mixture was concentrated, diluted with CH2Cl2, and filtered through a plug of silica gel. The solvent was removed under
vacuum and the product was purified by column chromatography (SiO2, petroleum ether/CH2Cl2=4:1) to give b1 (0.155 g, 78%) as a
1
colorless solid. H NMR (CDCl3, 500 MHz): δ = 0.99 (t, J=7.5 Hz, 12H), 1.38 (m, 8H), 1.62 (m, 8H), 3.31 (t, J=7.5 Hz, 8H), 6.59 (d,
J=8.5 Hz, 4H), 7.36 (d, J=8.5 Hz, 4H) ppm. FT-IR (KBr): υ=2947, 2874, 2200, 1587, 1527, 1441, 1367, 1024, 914, 804, 621, 523 cm-1.
MALDI-TOF-MS (dithranol): m/z: calc’d for C30H44N2: 432.35 g/mol, found: 433.3 g/mol [MH]+. Elemental analysis calc’d (%) for
C30H44N2 (432.69): C 83.28, H 10.25, N 6.47; found: C 83.27, H 10.26, N 6.47.
2.2.4. Synthesis of 2-(2-(4-(Dibutylamino)phenyl)-2-(4-(dicyanomethylene)-2,3,5,6-tetrafluorocyclohexa-2,5-dien-1-ylidene)-1-(4-
nitrophenyl)ethylidene)malononitrile (FTQ-2). The monomer b1 (120 mg, 0.361 mmol) and F4-TCNQ (0.090 g, 0.360 mmol) were
dissolved in chlorobenzene (2 mL). After, the reaction mixture was then stirred at 100 °C for 2 h under Ar atmosphere. The solvent was
removed under vacuum and the product was purified by column chromatography (SiO2, CH2Cl2) to give FTQ-2 (0.213 g, 97%) as a

black red solid. 1H NMR (CDCl3, 500 MHz): δ = 0.94 (t, J=7.5 Hz, 6H), 1.00 (t, J=7.5 Hz, 6H), 1.44 (m, 4H), 1.51 (m, 4H), 1.66 (m,
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4H), 1.78 (m, 4H), 3.57 (t, J=7.0 Hz, 4H), 3.61 (t, J=7.0 Hz, 4H), 6.62 (d, J=9.0 Hz, 2H), 6.83 (d, J=9.5 Hz, 2H), 7.41 (d, J=9.0 Hz, 2H),
7.56 (d, J=9.0 Hz, 2H) ppm. FT-IR (KBr): υ=2947, 2874, 2187, 1587, 1527, 1441, 1367, 1024, 914, 804, 621, 523 cm-1. MALDI-TOF-
MS (dithranol): m/z: calc’d for C42H44N6F4: 708.84 g/mol, found: 709.6 g/mol [MH]+. Elemental analysis calc’d (%) for C42H44N6F4
(708.84): C 71.17, H 6.26, N 11.86; found: C 71.19, H 6.28, N 11.87.
2.2.5. Synthesis of N,N-dibutyl-4-((4-pentylphenyl)ethynyl)aniline (b3). a4 (0.137 g, 0.600 mmol) and 1-bromo-4-pentylbenzene (0.113 g,
0.500 mmol) were dissolved in TEA (150 mL) and THF (150 mL). The solution was flushed with bubbling Ar gas for 30 min. Then the
Pd(PPh3)2Cl2 (12.6 mg, 0.0180 mmol) and CuI (5.7 mg, 0.030 mmol) were added. The reaction mixture was then stirred at 80 °C for 10
h under Ar atmosphere. The resulting mixture was concentrated, diluted with CH2Cl2, and filtered through a plug of silica gel. The
solvent was removed under vacuum and the product was purified by column chromatography (SiO2, petroleum ether/CH2Cl2=7:1) to
give b3 (0.135 mg, 60%) as a yellow oil liquid. 1H NMR (CDCl3, 500 MHz): δ = 0.94 (t, J=6.0 Hz, 3H), 1.00 (t, J=7.0 Hz, 6H), 1.39 (m,
8H), 1.68 (m, 6H), 2.65 (t, J=7.5 Hz, 2H), 3.33 (t, J=7.5 Hz, 4H), 6.62 (d, J=9.0 Hz, 2H), 7.17 (d, J=7.5 Hz, 2H), 7.40 (d, J=9.0 Hz, 2H),
7.45 (d, J=7.5 Hz, 2H) ppm. FT-IR (KBr): υ=2935, 2850, 2200, 1601, 1503, 1367, 1184, 112, 792, 731, 535 cm-1. MALDI-TOF-MS
(dithranol): m/z: calc’d for C27H37N: 375.29 g/mol, found: 376.2 g/mol [MH]+. Elemental analysis calc’d (%) for C27H37N (375.29): C
86.34, H 9.93, N 3.73; found: C 86.35, H 9.94, N 3.74.
2.2.6. Synthesis of 2-(2-(4-(Dibutylamino)phenyl)-2-(4-(dicyanomethylene)-2,3,5,6-tetrafluorocyclohexa-2,5-dien-1-ylidene)-1-(4-
pentylphenyl)ethylidene)malononitrile (FTQ-3). The monomer b2 (112 mg, 0.300 mmol) and F4-TCNQ (0.082 g, 0.30 mmol) were
dissolved in chlorobenzene (2 mL). The reaction mixture was then stirred at 100 °C for 2 h under Ar atmosphere. The solvent was
removed under vacuum and the product was purified by column chromatography (SiO2, CH2Cl2) to give FTQ-3 (0.19 g, 98%) as a
1
black red solid. H NMR (CDCl3, 500 MHz): δ = 0.92 (t, J=6.5 Hz, 3H), 1.04 (t, J=7.5 Hz, 6H), 1.36 (m, 4H), 1.48 (m, 4H), 1.67 (m,
2H), 1.75 (m, 4H), 2.68 (t, J=7.5 Hz, 2H), 3.60 (t, J=8.5 Hz, 4H), 6.86 (d, J=9.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 7.38 (d, J=9.0 Hz, 2H),
7.53 (d, J=8.0 Hz, 2H) ppm. FT-IR (KBr): υ=2935, 2850, 2200, 1601, 1379, 1012, 816, 645, 559 cm-1. MALDI-TOF-MS (dithranol):
m/z: calc’d for C39H37F4N5: 651.30 g/mol, found: 652.4 g/mol [MH]+. Elemental analysis calc’d (%) for C39H37F4N5 (651.30): C 71.87,
H 5.72, N 10.75; found: C 71.89, H 5.71, N 10.74.
3. Results and discussion
3.1. Synthesis of compounds FTQ-1~FTQ-4
The synthesis of donor-acceptor chromophores FTQ-1 to FTQ-4 was achieved through introducing amine-substituted aromatic
precursors by high-yielding [2+2] cycloaddition-cycloreversion reactions using F4-TCNQ as acceptor molecules. As shown in Scheme
1, all the acetylenic bonds reacted with the electron acceptor of F4-TCNQ. The reaction of F4-TCNQ could be fulfilled under ambient
temperature, while enhancing the temperature to 100 °C allowed for more rapid completion of the reaction. Furthermore, one easy
purification process was necessary because of the absence of byproducts, and yields were significantly high, ranging from 96% to 98%.
The molecular structures of FTQ were confirmed by NMR, FT-IR, and MS spectra and elemental analysis. The FTQ dyes were stable

at ambient conditions and reasonably soluble in common organic solvents, such as DMSO, CH2Cl2, THF, acetone and acetonitrile (Fig.
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S1).
The cycloaddition-cycloreversion reaction of b3 (~2×10-5 M) was initially investigated by UV/Vis/NIR spectroscopic titration
experiments by adding acceptor molecule F4-TCNQ to b3 in chlorinated solvents (Fig. 1a). The absorption intensities of the precursor
(b3) started to decrease, and two new charge transfer (CT) bands at 483 and 861 nm formed due to the presence of product FTQ-3. The
peak positions of the CT bands remained unchanged, while the intensities linearly increased with the addition of F4-TCNQ. Presence of
one isosbestic point at 357 nm indicates there were no side reactions. The UV/Vis/NIR monitoring indicates that the electronic state of
FTQ-3 was modulated by F4-TCNQ. Moreover, the two new CT bands of FTQ-3 have almost perfect geometrical shapes (Fig. 1b). It
is easy to separate the three peaks using software based on the Gaussian distribution, and the results are shown in Fig. 1b. Peaks A and
B usually exhibit Gaussian distribution and the fitting curves meet the experimental curves very well. This suggests that each peak
conceptually could represent one kind of chromophore. In accordance with the CT properties of every chromophore, the corresponding
chemical structures were also inserted into Fig. 1b.
3.2. PA effect of FTQ-1~FTQ-4
The PA intesnities of FTQ solutions with a concentration of 3 × 10-5 M were measured using MOST-128. As it is shown in Fig. 2a,
the PA intensities of all the FTQ dyes were higher than that of ICG, which was used as the archetypal reference for dye-based PA
contrast agents under the same experimental conditions. To give insight into the high PA intensities of FTQs and to cue the molecular
design of NIR dyes for PAI, we measured the molar extinction coefficients (ε), and the thermal conversion efficiencies (η) according to
the photothermal mechanism based equation of PA effect: q Γ η µa F (1), where Γ is the Grueneisen parameter (dimensionless), µa is
the optical absorption coefficient (cm-1), η is the thermal conversion efficiency, and F is the local optical fluence (J·cm-2). [15] The major
parameters for re-construction of the PA signals were the optical absorption coefficient (µa) and the thermal conversion efficiency (η).
Compounds FTQ-1 through FTQ-4 containing the same parent skeleton and different substitutions were used to measure the
UV/Vis/NIR absorption spectra at a concentration of 3 × 10-5 M (Fig. 2b). The UV/Vis/NIR absorption spectra of FTQ were in
accordance with the wavelength-dependent PA intensity diagrams (Fig. 2b and c), supporting the strong correlation between the NIR
absorption and PA effect. The parent molecule FTQ-1 showed the NIR absorption with the maxima at 817 nm and the molar extinction
coefficient of 2.2 × 104 L mol-1 cm-1. The compound FTQ-3 showed almost the same absorption properties as FTQ-1 (maxima at 816
nm, ε = 2.1 × 104 L mol-1 cm-1) with introduction of an alkyl chain. Interestingly, with the introduction of the electron-donating N,N-
dibutyl group as an auxochrome to FTQ-2, the molar extinction coefficient was increased to 4.5 × 104 L mol-1 cm-1 and the absorption
peak was hypsochromically shifted to 790 nm compared to the parent molecule FTQ-1. However, the placement of an electron-
withdrawing aldehyde group in the chromophore bathochromically shifted the NIR absorption peak of FTQ-4 to 831 nm compared to
FTQ-1, and the molar extinction coefficient increased to 3.8 × 104 L mol-1 cm-1. Based on the above experimental results, we can
conclude that the alkyl chains had almost no influence on the NIR absorption (wavelength and intensity). Electron-withdrawing groups
tended to bathochromically shift the absorption of the FTQ in the NIR range. On the contrary, electron-donating groups tended to

induce a hypsochromicshift of the absorption.AImCpCortEanPtlTy,EboDth MtheAelNecUtroSn-CdoRnaItPinTg and withdrawing groups resulted in increased
absorption intensity with a hypsochromic shift. [16] Based on the large molar extinction coefficients (ε), FTQ-2 and FTQ-4 showed PA
intensities 1.5 × 105 at 800 nm and 1.3 × 105 at 830 nm, respectively, which were higher than that of FTQ-1 (9.9 × 104 at 820 nm) and
FTQ-3 (4.2 × 104 at 820 nm). The results are in accordance with the equation (1) based on the photothermal mechanism.
In addition, thermal conversion efficiencies (η) of FTQ dyes were also determined in order to evaluate their contributions to the
resultant PA effects. The FTQ solutions with a concentration of 3 × 10-5 M were used to measure the thermal conversion efficiency. A
100 ꢀL solution of FTQ was irradiated under the laser (400 mW) at the wavelength of maximum absorption for each FTQ. The
temperatures were recorded by the thermal imaging system and the time-dependent temperature variation curves of FTQ are shown in
Fig. 2d. The thermal conversion efficiencies (η) of the FTQs were calculated by fitting the curves of time-dependent temperature
variations (Fig. S2). [17] FTQ-2 and FTQ-3 displayed high thermal conversion efficiencies of 51.5% and 53.9%, respectively (Table 1).
Comparing these with the relatively low thermal conversion efficiencies of FTQ-1 (η = 30.0%) and FTQ-4 (η = 33.9%), it is proposed
that the alkyl chains in FTQ-2 and FTQ-3 may contribute to the higher thermal conversion efficiencies due to the thermal vibration of
the alkyl chains, since they have similar molecular structures. [18] FTQ-3, with the highest η value (53.9%), showed the lowest PA
intensity (4.2 × 104 at 820 nm). This result was not supported by the photothermal equation (1), where the PA effect is directly related
to the thermal conversion efficiency. For the series of FTQ NIR dyes, the photoacoustic waves may be also related to the
electrostriction of the solvent, induced by charge transfer of the FTQ molecules. [19-21] The molecular simulation and quantum chemistry
calculations were carried out for explanation (Table 1 and details see SI). The possible conformational changes under photoirradiation
may contribute to the PA effect for the series of FTQs aside from the photothermal mechanism, and make the FTQ dyes show higher
PA intensities when compared to ICG. [22]
3.3. PA imaging in vitro
To demonstrate the potential application of the FTQs in PAI, we loaded FTQ-4 into a well-defined hydrophobic bilayer of liposomes,
[23]
which have been widely applied as drug delivery vehicles.
Briefly, L-α-phosphatidylcholine and cholesterol (4:1 w:w) were
dissolved in an ethanol solution. Afterward, the FTQ-4-containing DMSO solution was added into the aforementioned premixed
ethanol solution. The resulting mixture was subsequently injected into phosphate-buffered saline (PBS) and stirred for 1 h. The FTQ-4
molecules were preferentially embedded into a hydrophobic phospholipid bilayer of liposome through hydrophobic interactions. [24] The
morphology of FTQ-4 L in PBS (pH=7.4) was investigated by transmission electron microscopy (TEM). Under the mass ratio (1:10)
of FTQ-4 and phospholipids, uniform vesicular structures of FTQ-4 L with a size of 42 ± 18 nm were observed (Fig. 3b). The
technique of dynamic light scattering (DLS) measured a hydrodynamic diameter of FTQ-4
distribution (Fig. S4). The FTQ-4 L with a 1:10 mass ratio displayed almost the same absorption (λmax = 815 nm) with that of FTQ-4
in DMSO, indicating FTQ-4 was successfully loaded into the liposome (Fig. 3a). Furthermore, the FTQ-4 L was utilized as a
L of 79 ± 12 nm with narrow size
contrast agent for PA imaging in vitro by using human breast cancer MCF-7 cells as a model cell line. The MCF-7 cells grown on a
Petri dish (~107 cells) were incubated with FTQ-4 L (10 µM based on FTQ-4 molecules) and ICG (10 µM) separately for 2 h at 37

°C. The treated cells in PBS were mixed with aA2%CuCltErapPuTreEagDaroMse AsolNutiUonS(VC1R/VI2P=T1:1), and then added into the wells of an agarose
gel phantom made in advance. PA imaging was collected using Multi-Spectral Optoacoustic Tomography (MSOT) under an 815 nm
laser excitation at different time intervals. The PA signals of FTQ-4 L in the cells were recorded in an agarose gel for PAI. About
50% of the PA signals could be maintained in FTQ-4 L treated cells for 96 h. As a reference, it only took 12 h for PA signals of ICG
treated cells to decrease by half (Fig. 3c). These results indicate that FTQ-4 could be used as a contrast agent for PAI when doping into
the nano-sized liposomes. In addition, the cytotoxicity of FTQ-4 L nanovesicles was evaluated through CCK-8 assays to determine
the metabolic viability of MCF-7 cells.[25] No significant cytotoxicity was observed with a concentration up to 10 µM under our
experimental conditions (Fig. S5).
4. Conclusions
In summary, we have prepared a series of NIR absorbing dyes through a cycloaddition-cycloreversion reaction. The systematic
investigation of the NIR dyes indicates that substitutions with electron-donating or electron-withdrawing groups can enhance the molar
extinction coefficients and PA intensities. Finally, the NIR absorbing dyes with good stability and high PA intensities were utilized to
image cells in an agarose gel for an extended period of time by loading them into nano-sized liposomes. Our results systematically
illuminate the influences on PA effects and provide guidance for the design of NIR absorbing dyes for high-performance PA
bioimaging. The investigation into water-soluble NIR absorbing dyes with high PA intensities is in progress in our lab.
Acknowledgements
This work was supported by National Natural Science Foundation of China (51473188, 21374026, 21304023, and 51303036),
National Basic Research Program of China (973 Program, 2013CB932701), Beijing Natural Science Foundation (2132053) and the
100-Talent Program of the Chinese Academy of Science (Y2462911ZX).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/XXX
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ACCEPTED MANUSCRIPT
Scheme 1. Molecular structures of compounds a, b and FTQ and their synthetic routes: (i) C4H9I, K2CO3, DMF, 100 °C, 12 h. (ii) Ethynyltrimethylsilane,
TEA/THF, Pd(PPh3)2Cl2, CuI, 40
°C, 12 h. (iii) K2CO3, CH3OH/THF. (iv) 4-Bromophenyl-R1, TEA/THF, Pd(PPh3)2Cl2, CuI, 80 °C, 12 h. (v) F4-
TCNQ, chlorobenzene, 100 C, 2 h
°
.

Fig. 1. (a) UV/Vis/NIR spectral changes of b3 (~2×10-5 M) upon titration with F4-TCNQ (0-1.0 equiv.) in chlorinated solvent at 100 °C. (b) The FTQ-3
ACCEPTED MANUSCRIPT
UV/Vis/NIR was fitted based on based on the Gaussian distribution, A Peak in blue, B peak in red and C peak in dark green.
Fig. 2. (a) The PA intensities of FTQ and ICG measured in DMSO (3 × 10-5 M). (b) The UV/Vis/NIR spectra of the NIR dyes FTQ and (c) corresponding photos
of FTQ solutions taken by digital camera. (d) The heating/cooling curves of FTQ at different times. The energy input from lasers was 400 mW at about 800 nm
with an irradiated diameter of 2 mm.

ACCEPTED MANUSCRIPT
Table 1. The summary of PA intensity, thermal conversion efficiency (η), molar extinction coefficient (ε), and corresponding sum of main intramoleular
interaction energy (V(r)) in both states of FTQ molecules.
V(r) (KJ/mol)
PA (104) ε (104, L/mol/cm)
FTQ
η
(%)
S1
S2
9.9
14.6
4.2
2.2
4.5
2.1
3.8
30.0 -90.485 -78.611
51.5 -107.156 -105.537
53.9 -89.996 -78.716
33.9 -90.073 -78.223
1
2
3
4
13.2

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Fig. 3. UV/Vis/NIR absorption spectra of FTQ-4 L with different ratios of FTQ-4 to liposome in PBS. (b) TEM images of FTQ-4 L. (c) PA imaging of
FTQ-4 L incubated in cells, which was added to agarose gel wells and embedded into the agarose gel phantom. The phantom was imaged using MOST 128
with an 815 nm excitation pulse laser at different time intervals.

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ꢀ A new class of dyes with NIR absorption was designed and prepared.
ꢀ The NIR dyes showed excellent photoacoustic effect.
ꢀ Charge transfer of NIR dyes may contribute to photoacoustic effect.