Heavy atom enhanced generation of singlet oxygen in novel indenofluorene-based two-photon absorbing chromophores for photodynamic therapy

Article abstract of DOI:10.1016/j.dyepig.2015.01.019

A series of indenofluorene-based two-photon absorbing chromophores (FL0, FL-2BrN, FL-1IN, FL-2IN, and FL-2IS) have been designed and synthesized for photodynamic therapy (PDT). Two-photon absorption (TPA) properties of these chromophores are determined and their structure-property relationship has been established. By incorporating electron withdrawing halogen atoms (such as Br, I) into the π-conjugation backbone of the chromophores, enhanced TPA cross-section values up to 2707 GM were achieved due to the formation of a donor-acceptor-donor (D-A-D) motif. At the same time, upon excitation at 430 nm, 2.2-4.1 times of enhancement in the singlet oxygen generation yield was observed for the photosensitizers with the heavy atoms incorporated. The photosensitizers with heavy atoms also show increased singlet oxygen generation efficiencies upon two-photon excitation at 808 nm, compared to those photosensitizers without heavy atoms. Photocytotoxicity of FL-2IN against cancer cells via two-photon excitation (808 nm) demonstrated that the indenofluorene-based chromophores are effective singlet oxygen donors for two-photon PDT.

Full text of DOI:10.1016/j.dyepig.2015.01.019

Dyes and Pigments 117 (2015) 7e15
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Heavy atom enhanced generation of singlet oxygen in novel
indenofluorene-based two-photon absorbing chromophores for
photodynamic therapy
1
1
1
1
*
Changquan Tang , Ping Hu , En Ma , Mingdong Huang , Qingdong Zheng
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 155 Yangqiao West Road,
Fuzhou 350002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 December 2014
Received in revised form
A series of indenofluorene-based two-photon absorbing chromophores (FL0, FL-2BrN, FL-1IN, FL-2IN,
and FL-2IS) have been designed and synthesized for photodynamic therapy (PDT). Two-photon ab-
sorption (TPA) properties of these chromophores are determined and their structure-property rela-
tionship has been established. By incorporating electron withdrawing halogen atoms (such as Br, I) into
17 January 2015
Accepted 20 January 2015
Available online 3 February 2015
the
p-conjugation backbone of the chromophores, enhanced TPA cross-section values up to 2707 GM
were achieved due to the formation of a donor-acceptor-donor (D-A-D) motif. At the same time, upon
excitation at 430 nm, 2.2e4.1 times of enhancement in the singlet oxygen generation yield was observed
for the photosensitizers with the heavy atoms incorporated. The photosensitizers with heavy atoms also
show increased singlet oxygen generation efficiencies upon two-photon excitation at 808 nm, compared
to those photosensitizers without heavy atoms. Photocytotoxicity of FL-2IN against cancer cells via two-
photon excitation (808 nm) demonstrated that the indenofluorene-based chromophores are effective
singlet oxygen donors for two-photon PDT.
Keywords:
Two-photon absorption
Photodynamic therapy
Singlet oxygen
Heavy atom effect
Cytotoxicity
Photosensitizer
©
2015 Elsevier Ltd. All rights reserved.
1. Introduction
frequency (long-wavelength) photons. With two-photon excita-
tion, near IR light at 750e1000 nm where tissue is more trans-
1
In the past two decades, photodynamic therapy (PDT) has been
established as a valid clinical treatment method for cancer and
other diseases [1e3]. In PDT, a photosensitizer is excited to its
singlet excited state by the absorption of a photon, and decays to its
triplet excited state via intersystem crossing (ISC). The photosen-
parent can be used to activate the photosensitizers for
O
2
generation [12]. Besides a deep tissue penetration, another
advantage of two-photon PDT is that the treatment is localized at
the focal point of a laser beam because the TPA process quadrati-
cally depends on the laser intensity, which provides a more precise
sitizer in the triplet state can subsequently transfer its energy to the
treatment [13]. The key prerequisite for two-photon PDT is that the
3
1
surrounding oxygen in the ground state ( O
2
), resulting in gener-
photosensitizers should efficiently generate O
2
under two-photon
1
ation of singlet oxygen ( O
2
) [4,5]. Singlet oxygen is a highly reac-
excitation. To obtain more efficient two-photon photosensitizers,
much effort have been devoted to improve TPA cross-sections of
traditional photosensitizers (such as porphyrins) via conjugating
peripheral energy donors with large TPA cross sections [14e16], or
to synthesize new photosensitizers with large TPA cross sections
[17e20].
tive oxygen species that can damage biological cell components
such as lipids [6], nucleic acids [7,8], or proteins [9,10]. In current
PDT, one of the main problems is the limited penetration of visible
light into tissue, restricting PDT to the treatment of ailments on
tissue surface [11].
Two-photon absorption (TPA) is a nonlinear process where a
molecule is promoted to its excited states by absorbing two low-
A molecule with large TPA generally should have an extended p-
conjugation system with good planarity, and strong electron ac-
ceptors and (or) donors attached in the terminals because the TPA
cross section value for a given molecule is related to the extent of
intramolecular charge transfer of the molecule upon photoexcita-
tion. Ladder-type oligo-p-phenylenes are good building blocks to
construct materials with large TPA cross-section values owning to
*
Corresponding author. Tel./fax: þ86 591 83721625.
E-mail address: qingdongzheng@fjirsm.ac.cn (Q. Zheng).
Tel./fax: þ86 591 83721625.
1
http://dx.doi.org/10.1016/j.dyepig.2015.01.019
143-7208/© 2015 Elsevier Ltd. All rights reserved.
0

8
C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
their planar chemical structures with extended
p
-conjugation
[33,34], and 8 [35,36] were prepared according to the literature
[
21,22]. In our previous reports, the relationship between TPA
procedures. Column chromatography was conducted with silica gel
1
13
properties of ladder-type oligo-p-phenylene-cored chromophores
60 (200e300 mesh). H NMR and C NMR spectra were recorded at
400 and 100 MHz, respectively. The MALDI-TOF spectra were
recorded on a Voyager-DE STR mass spectrometer. Absorption and
fluorescence spectra were acquired using a spectrometer (Lambda
35 UV/vis) and a Cary fluorimeter, respectively. For nonlinear op-
tical experiments, the excitation pulse (1 KHz, 240e2600 nm,
pulse-width <120 fs) was generated from an optical parametric
amplifier (TOPAS-F-UV2, Spectra-Physics) pumped by a regener-
atively amplified femtosecond Ti-sapphire laser system (800 nm,
1 KHz, pulse energy 4 mJ, pulse-width <120 fs, Spitfire Pro-F1KXP,
Spectra-Physics), which is seeded by femtosecond Ti-sapphire
oscillator (80 MHz, pulse-width <70 fs, 710e920 nm, Maitai XF-1,
Spectra-Physics). The one-photon excitation singlet oxygen emis-
sion was measured by using a FLS980 spectrofluorometer. The
pump source for two-photon excitation as well as TPA cross-section
measurements was generated by a mode locked Ti:sapphire laser.
The laser beam was focused by an f ¼ 10 cm lens, and the solution
sample in a 1 cm fluorimeter cuvette (four optically clear windows)
was placed at a fixed distance of ~11.5 cm from the focusing lens.
Long-pass filter was used to attenuate the excitation laser beam.
and their conjugation lengths was systematically investigated
[
23,24]. It was found that the TPA cross section values of the ladder-
type oligo-p-phenylenes-cored chromophores increase with the
increasing -conjugation lengths of the central ladder-type oligo-
p
p-phenylene core. On the other hand, it has been found that pro-
moting an ISC rate is an efficient method to improve the singlet
oxygen generation of a chromophore. The rate of ISC can be
enhanced by strong spin-orbit coupling in the presence of heavy
atoms (such as I, Br) which are incorporated into the chromophore
or are external but close enough to the chromophore, termed as a
heavy atom effect [25e27]. On the basis of these considerations, we
design a series of indenofluorene-based chromophores with heavy
halogen atoms incorporated and anticipate that the introduction of
heavy halogen atoms into the chromophores will lead to promising
1
photosensitizers with large TPA cross sections and high O
2
gen-
eration rates. Although the two-photon properties of
indenofluorene-based chromophores have been investigated
1
[
28e30], their O
2
generation properties under two-photon exci-
tation are still unexplored. Hence, the heavy atom effect on the TPA
1
as well as O
knowledge.
2
generation has never been reported to the best of our
2.2. Methods
In this paper, we report on the design and synthesis of a series of
novel indenofluorene-based chromophores as two-photon PDT
photosensitizers by incorporating heavy halogen atoms (Chart 1).
The linear and TPA properties, as well as the one- and two-photon
excited fluorescence properties for these chromophores were
investigated. Singlet oxygen generation efficiencies for these pho-
tosensitizers under one- and two-photon excitation have also been
evaluated. The application of chromophore FL-2IN as a photosen-
sitizer for two-photon photodynamic therapy was successfully
demonstrated.
2.2.1. Cytotoxicity assays
The MCF-7 cells (human breast carcinoma) were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% FBS (fetal bovine serum) in an atmosphere of 5% CO and 95%
2
ꢀ
air at 37 C. Immediately before the experiments, the cells (8000
per well) were placed in a 96-multiwell plate, followed by addition
of increasing concentrations of compound FL-2IN (99% DMEM and
1% DMSO). The photosensitizer solutions were prepared by
dispersing the DMSO solution of FL-2IN to the DMEM solution
directly to form nanoparticles. The final concentrations of the
2
. Experimental section
photosensitizer were 0e40
bated at 37 C in an atmosphere of 5% CO
24 h, followed by MTT assays.
m
M (n ¼ 4). The cells were then incu-
ꢀ
ꢀ
2
and 95% air at 37 C for
2.1. Materials and instruments
Reagents were purchased from Aldrich Inc. and Adamas-beta
2.2.2. Two-photon PDT against cancer cells
Chemical Ltd. and used without further purification unless other-
wise stated. The starting materials 1 [23], 3 [31], 4 [31], 6 [32], 7
MCF-7 cancer cells were placed in 96-multiwell plates about
8000 per well and incubated overnight at 37 C in a humidified
ꢀ
C H
6 13
C H
6 13
C
6
H
13
6 13
C H
N
N
6 13
C H
6 13
C H
FL0
C
6
H
13
C H
6 13
C
6
H
13
6 13
C H
N
R
2
C H
6 13
6 13
C H
C
6
H
13
C
6
H
13
R
1
N
FL-2BrN: R
FL-1IN: R
FL-2IN: R
1
1
1
= R
= I, R
= R = I
2
= Br
2
C
6
H
13
6 13
C H
= H
C H
6 13
6 13
C H
S
2
I
C
6
H
13
6 13
C H
6 13
C H
6 13
C H
I
FL-2IS
S
C H
6 13
6 13
C H
Chart 1. Molecular structures of indenofluorene-based chromophores FL0, FL-2BrN, FL-1IN, FL-2IN, and FL-2IS.

C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
9
incubator with 5% CO
removed, and the cells were treated with the medium (200
2
atmosphere. The DMEM medium was
L)
2.3.4. Synthesis of compound 10
m
This compound was prepared using a similar procedure to that
containing FL-2IN at various concentrations. After incubation 8 h,
cells were washed using sterile PBS and added fresh medium. Each
well was exposed to laser irradiation at 808 nm with power density
of ~3.0 W cm . Then the cells were further incubated for 12 h
followed by the MTT colorimetric assay. Cell experiments without
photosensitizer under the same experimental conditions were
performed as controls.
used for the synthesis of 5. Quantities: compound 9 (0.60 g,
0.61 mmol), compound 4 (0.16 g, 1.21 mmol), Pd(OAc)
0.05 mmol), P(o-tolyl) (61 mg, 0.2 mmol), Et N (1.0 ml), CH
(20 ml). The title compound was obtained as a yellow crystalline
2
(12 mg,
3
3
3
CN
ꢁ
2
1
ꢀ
solid (0.33 g, 52%). H NMR (400 MHz, CDCl
3
, 25 C, TMS):
d
¼ 10.00
(s, 1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.77e7.66 (m, 4H), 7.61e7.47 (m,
10H), 7.45e7.36 (m, 2H), 7.30 (d, J ¼ 8.4 Hz, 3H), 7.22e7.16 (m, 3H),
7
.09 (s, 2H), 2.94 (t, J ¼ 7.6 Hz, 2H), 2.12e1.96 (m, 8H), 1.73e1.66 (m,
2
2
.3. Synthesis
2H), 1.48e1.40 (m, 2H), 1.37e1.23 (m, 7H), 1.17e0.96 (m, 24H),
0
.91e0.63 (m, 20H); MS (MALDI-TOF, m/z): calcd for C75
H92OS,
þ
.3.1. Synthesis of compound 2
[M ] 1040.6; found, 1040.7.
A mixture of compound 1 (1.0 g, 1.34 mmol), diphenylamine
(
0.23 g, 1.34 mmol), and sodium t-butoxide (0.15 g, 1.56 mmol) in
2.3.5. Synthesis of compound FL0
dry toluene (40 ml) was stirred at room temperature for 30 min
under nitrogen atmosphere. Then tri(dibenzylideneacetone)dipal-
ladium(0) (18 mg, 0.027 mmol) and 1,1’-bis(diphenylphosphino)
ferrocene (36 mg, 0.054 mmol) were added to the mixture. The
mixture was heated to reflux for 12 h and then cooled to room
temperature, diluted with diethyl ether (60 ml), and washed with
brine and water in sequence. The resulting organic layer was dried
over sodium sulfate and concentrated in vavuo to give the crude
product, and it was purified by column chromatography (eluent:
This compound was prepared using a similar procedure to that
used for the synthesis of 5. Quantities: compound 2 (0.80 g,
0.96 mmol), compound 3 (50 mg, 0.38 mmol), Pd(OAc)
0.05 mmol), P(o-tolyl) (61 mg, 0.2 mmol), Et N (1.0 ml), CH
(20 ml). The title compound was obtained as a yellow crystalline
2
(12 mg,
3
3
3
CN
1
ꢀ
solid (0.29 g, 46%). H NMR (400 MHz, CDCl
3
, 25 C, TMS):
d
¼ 7.70
(d, J ¼ 7.6 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 2H), 7.60e7.48 (m, 12H),
7.29e7.17 (m, 12H), 7.12 (d, J ¼ 7.6 Hz, 10H), 7.06 (d, J ¼ 8.0 Hz, 2H),
7.02e6.98 (m, 4H), 2.10e1.82 (m, 16H), 1.12e0.83 (m, 48H),
13
ꢀ
ethyl acetate/petroleum ether ¼ 1/40) on silica gel to afford the title
0.81e0.56 (m, 40H); C NMR (100 MHz, CDCl , 25 C, TMS):
3
1
compound as a yellow oil (0.50 g, 45%). H NMR (400 MHz, CDCl
3
,
d
¼ 152.65, 151.64, 150.52, 150.12, 148.08, 146.87, 141.55, 140.46,
ꢀ
2
5 C, TMS):
d
¼ 7.61 (d, J ¼ 8.4 Hz, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 7.51
139.71, 136.90, 136.83, 135.83, 129.34, 129.17, 127.23, 126.81, 125.67,
123.90, 123.65, 122.38, 120.68, 120.13, 119.83, 119.54, 113.84, 113.40,
(
(
(
d, J ¼ 7.6 Hz, 2H), 7.44 (d, J ¼ 7.2 Hz, 2H), 7.23 (d, J ¼ 7.2 Hz, 4H), 7.11
d, J ¼ 7.6 Hz, 5H), 7.07e6.97 (m, 3H), 2.02e1.84 (m, 8H), 1.18e0.95
54.78, 54.72, 40.90, 40.58, 31.62, 31.59, 29.82, 29.68, 23.81, 22.62,
þ
m, 24H), 0.79e0.55 (m, 20H); MS (MALDI-TOF, m/z): calcd for
22.56, 14.07. MS (MALDI-TOF, m/z): calcd for C111
1641.1; found, 1641.9. Elemental analysis: calcd for C111
9.21, H 9.08, N 1.71; found: C 89.52, H 8.93, N 1.95.
H
147
S
2
, [M ]
þ
C
56
H
70BrN, [M ] 835.4; found, 835.5.
147 2
H S : C
8
2.3.2. Synthesis of compound 5
Compound 2 (0.5 g, 0.6 mmol), compound 4 (0.1 g, 0.76 mmol),
2.3.6. Synthesis of compound FL-2BrN
Pd(OAc)
2
(12 mg, 0.05 mmol), P(o-tolyl)
3
(61 mg, 0.2 mmol), Et
3
N
To compound 6 (68 mg, 0.126 mmol) in dry THF (30 ml), was
added sodium tert-butoxide (0.146 g, 1.52 mmol). The reaction
mixture was cooled to 0 C in an ice bath. Compound 5 (0.26 g,
0.29 mmol) was added to the solution, the ice bath was removed
and the mixture was stirred at room temperature for 12 h. The
reaction mixture was poured into 100 ml of water and then THF
was removed. The crude product was collected by filtration and
further purified by column chromatography with ethyl acetate/
petroleum ether (1/40), affording the title compound as a yellow
(
1.0 ml), CH
3
CN (20 ml) were added to a pressure tube with a
ꢀ
plunger valve and a magnetic bar under argon. The resulting
mixture was heated to reflux for 24 h and then cooled to room
temperature. The mixture was poured into methanol (50 ml) with
vigorous stirring. The precipitate formed was collected on a filter
funnel and washed thoroughly with methanol, and the crude
product was purified by column chromatography (eluent: ethyl
acetate/petroleum ether ¼ 1/15) on silica gel to afford the title
1
ꢀ
3
, 25 C,
compound as a yellow solid. The title compound was obtained as a
yellow crystalline solid (0.26 g, 49%). H NMR (400 MHz, CDCl
crystalline solid (0.19 g, 77%). H NMR (400 MHz, CDCl
TMS):
1
3
,
d
¼ 7.91 (s, 2H), 7.71 (d, J ¼ 8.0 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 2H),
ꢀ
2
3
1
3
5 C, TMS):
d
¼ 10.00 (s, 1H), 7.89e7.84 (m, 2H), 7.76e7.68 (m,
7.61e7.56 (m, 14H), 7.52 (d, J ¼ 18.4 Hz, 2H), 7.41 (d, J ¼ 15.6 Hz, 2H),
7.35e7.18 (m, 12H), 7.17e7.10 (m, 12H), 7.07e7.06 (m, 2H),
7.05e6.98 (m, 4H), 2.12e1.85 (m, 16H), 1.20e0.96 (m, 48H),
H), 7.61 (d, J ¼ 7.6 Hz, 1H), 7.58e7.51 (m, 5H), 7.39 (d, J ¼ 8.4 Hz,
H), 7.23 (d, J ¼ 7.6 Hz, 4H), 7.12 (d, J ¼ 7.6 Hz, 5H), 7.17e6.98 (m,
13
ꢀ
3
, 25 C, TMS):
H), 2.08e1.86 (m, 8H), 1.17e0.95 (m, 24H), 0.81e0.54 (m, 20H);
0.88e0.56 (m, 40H); C NMR (100 MHz, CDCl
¼ 152.63, 151.66, 150.52, 150.11, 148.05, 146.87, 141.72, 140.50,
139.63, 137.92, 136.85, 135.70, 135.61, 131.88, 130.32, 129.99, 129.16,
27.44, 126.95, 126.84, 125.77, 125.40, 123.87, 123.64, 123.13, 122.37,
þ
MS (MALDI-TOF, m/z): calcd for C65
H77NO, [M ] 887.6; found,
d
8
87.6.
1
2.3.3. Synthesis of compound 9
120.73, 120.13, 119.80, 119.55, 113.86, 113.38, 54.76, 54.72, 40.87,
This compound was prepared using a similar procedure to that
used for the synthesis of 5. Quantities: compound 1 (0.748 g,
.0 mmol), (E)-hexyl(4-(4-vinylstyryl)phenyl)sulfane (0.28 g,
40.55, 31.60, 31.57, 29.80, 29.65, 23.79, 22.60, 22.54, 14.06, 14.04.
þ
MS (MALDI-TOF, m/z): calcd for C138
2
H158Br N
2
, [M ] 2004.5; found,
1
2004.1. Elemental analysis: calcd for C138
H
158Br
2
N
2
: C 82.69, H 7.94,
0
.87 mmol), Pd(OAc)
2
3
(12 mg, 0.05 mmol), P(o-tolyl) (61 mg,
N 1.40; found: C 82.75, H 8.01, N 1.26.
0
.2 mmol), Et N (1.0 ml), CH
3
3
CN (20 ml). The title compound was
1
obtained as a yellow solid (0.56 g, 57%). H NMR (400 MHz, CDCl
3
,
2.3.7. Synthesis of compound FL-1IN
ꢀ
2
5
C, TMS):
d
¼ 7.71 (d, J ¼ 8.4 Hz, 1H), 7.68e7.56 (m, 4H),
This compound was prepared using a similar procedure to that
used for the synthesis of FL-2BrN. Quantities: compound 7 (70 mg,
0.14 mmol), sodium tert-butoxide (0.16 g, 1.67 mmol), compound 5
7
7
8
1
.55e7.46 (m, 4H), 7.45e7.37 (m, 5H), 7.30 (d, J ¼ 8.4 Hz, 2H),
.22e7.13 (m, 2H), 7.08 (s, 2H), 2.94 (t, J ¼ 7.6 Hz, 2H), 2.10e1.92 (m,
H), 1.71e1.63 (m, 2H), 1.51e1.37 (m, 4H), 1.35e1.21 (m, 5H),
.12e0.83 (m, 24H), 0.81e0.56 (m, 20H); MS (MALDI-TOF, m/z):
(0.30 g, 0.34 mmol), THF (40 ml). The title compound was obtained
1
as a yellow crystalline solid (0.18 g, 64%). H NMR (400 MHz, CDCl
3
,
þ
ꢀ
calcd for C66
H
85BrS, [M ] 988.5; found, 988.6.
25 C, TMS):
d
¼ 8.11 (d, J ¼ 7.6 Hz, 2H), 7.89 (d, J ¼ 8.4 Hz, 2H),

10
C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
7
7
.77e7.66 (m, 4H), 7.63 (d, J ¼ 8.4 Hz, 4H), 7.60e7.45 (m, 12H),
.43e7.39 (m, 2H), 7.29e7.17 (m,10H), 7.16e7.11 (m,10H), 7.09e7.07
m, 2H), 7.05e6.98 (m, 6H), 2.12e1.82 (m, 16H), 1.18e0.96 (m, 48H),
divinylbenzene 3 in 46% yield. Compounds FL-2BrN, FL-1IN, FL-
2IN, and FL-2IS were obtained in 64e88% yields by two-fold Hor-
nereEmmonseWittig coupling reactions of the phosphonates (6, 7,
or 8) with an excess amount of the corresponding aldehydes (5 and
(
0
d
.84e0.52 (m, 40H); 13C NMR (100 MHz, CDCl
¼ 152.64, 151.66, 150.53, 150.13, 148.08, 146.88, 141.66, 140.49,
, 25 C, TMS):
ꢀ
3
ꢀ
10) using sodium tert-butoxide as a base in THF at 0 C. The inter-
1
1
1
1
39.66, 139.12, 138.42, 137.65, 137.57, 137.50, 136.88, 136.12, 136.02,
35.70, 131.75, 131.00, 129.77, 129.52, 129.15, 127.27, 127.07, 126.83,
26.41, 126.25, 125.98, 125.74, 123.88, 123.65, 122.37, 120.72, 120.11,
19.83, 119.53, 113.85, 113.39, 54.77, 54.72, 40.85, 40.54, 31.59,
1.56, 29.79, 29.65, 23.80, 22.59, 22.53, 14.03, 14.01. MS (MALDI-
mediate aldehydes (5 and 10) were synthesized by reacting com-
pounds 2 and 9 with 4-vinylbenzaldehyde (4) via Heck reactions in
49% and 52% yields, respectively. One-fold amination of compound
1 with diphenylamine afforded compound 2 in 45% yield. Com-
pound 9 was obtained by a one-fold Heck reaction between com-
pound 1 and (E)-hexyl(4-(4-vinylstyryl)phenyl)sulfane in 57% yield.
3
þ
TOF, m/z): calcd for C138
Elemental analysis: calcd for C138
found: C 84.19, H 8.11, N 1.12.
H
159IN
2
, [M ] 1972.6; found, 1972.2.
: C 84.02, H 8.12, N 1.42;
1
13
H
159IN
2
All new compounds were characterized by H NMR, C NMR,
MALDI-TOF MS, and the purity of the target chromophores were
further confirmed by elemental analysis.
2
.3.8. Synthesis of compound FL-2IN
This compound was prepared using a similar procedure to that
used for the synthesis of FL-2BrN. Quantities: compound 8 (85 mg,
.13 mmol), sodium tert-butoxide (0.16 g, 1.67 mmol), compound 5
0.30 g, 0.34 mmol), THF (40 ml). The title compound was obtained
3.2. Linear absorption and emission
0
(
The linear absorption and emission spectra for compounds FL0,
FL-2IS, FL-1IN, FL-2IN, and FL-2BrN in THF are shown in Fig. 1, and
the corresponding data are summarized in Table 1. As shown in
Fig. 1, the linear absorption bands for these compounds were
similarly located around 430 nm, and a bathochromic shift was
observed in going from FL0 (422 nm) to FL-2IS (423 nm), to FL-1IN
(424 nm), to FL-2IN (430 nm), and to FL-2BrN (434 nm). This trend
in absorption for the chromophores is due to the negative inductive
effect of the halogen atoms in the chromophore molecules and the
1
as a yellow crystalline solid (0.24 g, 88%). H NMR (400 MHz, CDCl
2
3
,
ꢀ
5
C, TMS):
d
¼ 8.07 (s, 1H), 7.70 (d, J ¼ 8.0 Hz, 2H), 7.60 (d,
J ¼ 8.0 Hz, 4H), 7.59e7.48 (m, 16H), 7.38 (d, J ¼ 17.6 Hz, 2H),
.29e7.16 (m, 10H), 7.14e7.10 (m, 12H), 7.05e7.02 (m, 2H),
.01e6.97 (m, 6H), 2.12e1.84 (m, 16H), 1.19e0.97 (m, 48H),
7
7
.81e0.56 (m, 40H); 13C NMR (100 MHz, CDCl
¼ 152.64, 151.75, 151.66, 150.53, 150.12, 148.06, 146.88, 143.86,
41.72, 140.80, 140.51, 139.64, 137.88, 136.86, 136.71, 136.38, 136.32,
36.13, 135.64, 135.11, 132.01, 131.90, 130.37, 130.32, 130.10, 129.97,
29.17, 127.43, 127.20, 126.98, 126.85, 126.76, 126.71, 125.79, 123.88,
23.69, 123.65, 122.38, 120.73, 120.14, 119.81, 119.56, 113.87, 113.40,
, 25 C, TMS):
ꢀ
0
3
d
1
1
1
1
extended
no halogen atom and a shorter
sorption maximum at 422 nm, while the absorption maximum of
compound FL-2IN with two iodine atoms and a longer -conju-
gation length shifts to 430 nm. Compound FL-2BrN has the same
p
-conjugation length. For example, compound FL0 with
p
-conjugation length has an ab-
p
5
2
C
4.77, 54.73, 40.87, 40.56, 31.61, 31.58, 29.81, 29.78, 29.74, 29.66,
p-
3.80, 22.62, 22.55, 14.07, 14.06. MS (MALDI-TOF, m/z): calcd for
conjugation length as compound FL-2IN, whereas the former
compound has a red-shifted absorption maximum of 434 nm, due
to the stronger negative inductive effect of the bromine atom. On
the other hand, the number of halogen atoms in the chromophore
molecules also affects their absorption maxima. As an example,
chromophore FL-2IN with two iodine atoms has a red-shifted ab-
sorption maximum compared to that of chromophore FL-1IN with
one iodine atom. For the emission spectrum of compound FL0,
there were two emission peaks, at around 477 nm and 494 nm.
However, only one emission peak was found for compounds FL-2IS,
FL-1IN, FL-2IN, and FL-2BrN. This difference may be due to the
more nonpolar nature of compound FL0 without halogen atoms,
which may disturb the solvent-chromophore interactions. The
emission maxima of the chromophores follow the same trend as
that observed in their absorption maxima. As listed in Table 1, the
emission peaks for the chromophores follow this order: FL0
(472 nm) < FL-2IS (491 nm) < FL-1IN (518 nm) < FL-2IN
(543 nm) < FL-2BrN (552 nm). Similarly, the same trend was also
found in the Stokes shifts of these chromophores, which follows the
same order: FL0 < FL-2IS < FL-1IN < FL-2IN < FL-2BrN. For
þ
H
138 158
I
2
N
2
, [M ] 2098.5; found, 2098.4. Elemental analysis: calcd
158 2 2
for C138H I N : C 78.98, H 7.59, N 1.33; found: C 79.24, H 7.69, N
1.04.
2.3.9. Synthesis of compound FL-2IS
This compound was prepared using a similar procedure to that
used for the synthesis of FL-2BrN. Quantities: compound 8 (85 mg,
.13 mmol), sodium tert-butoxide (0.16 g,1.67 mmol), compound 10
0.33 g, 0.31 mmol), THF (40 ml). The title compound was obtained
0
(
1
as a yellow crystalline solid (0.26 g, 83%). H NMR (400 MHz, CDCl
3
,
ꢀ
2
5 C, TMS):
d
¼ 8.12 (s, 2H), 7.79e7.72 (m, 4H), 7.69e7.58 (m, 12H),
7.57e7.49 (m, 10H), 7.45e7.40 (m, 6H), 7.39e7.29 (m, 10H),
7
.25e7.16 (m, 5H), 7.09 (d, J ¼ 5.6 Hz, 3H), 7.05e6.98 (m, 2H), 2.94 (t,
J ¼ 7.6 Hz, 4H), 2.12e1.93 (m, 16H), 1.71e1.64 (m, 4H), 1.49e1.40 (m,
4
0
d
H), 1.38e1.25 (m, 8H), 1.17e0.95 (m, 48H), 0.93e0.84 (m, 6H),
.81e0.52 (m, 40H); 13C NMR (100 MHz, CDCl
¼ 151.77, 151.56, 150.57, 150.38, 141.59, 141.39, 140.78, 140.39,
37.87, 137.70, 137.01, 136.32, 135.87, 135.71, 134.91, 132.01, 130.89,
30.61,130.32, 130.13,129.88, 129.50, 129.36,129.26, 128.88, 128.54,
27.95, 127.71, 127.20, 126.96, 126.53, 125.81, 124.70, 123.34, 120.82,
19.73, 119.42, 114.07, 113.98, 100.46, 54.87, 54.77, 40.79, 33.56,
1.47, 31.39, 30.24, 29.76, 29.68, 29.14, 28.57, 28.43, 23.79, 22.57,
, 25 C, TMS):
ꢀ
3
1
1
1
1
ꢁ1
instance, compound FL0 has a Stokes shift of 2510 cm , whereas
ꢁ
1
the Stokes shift value for compound FL-2BrN is 4925 cm . This
increased Stokes shift for the compound with a halogen atom can
be attributed to its more polar nature which somewhat improves
the solute/solvent interactions.
3
2
2.51, 22.43, 22.23, 14.02. MS (MALDI-TOF, m/z): calcd for
þ
C
H
158 188
I S
2 2
, [M ] 2405.1; found, 2405.6. Elemental analysis: calcd
for C158
H
188
I
2
S
2
: C 78.90, H 7.88; found: C 78.64, H 7.72.
As shown in Table 1, the molar extinction coefficients of each
compound in THF are relatively large with a range from
1.43 ꢂ 10 M cm to 2.52 ꢂ 10 M cm , and the values follow
this order: FL0 > FL-2BrN > FL-1IN > FL-2IS > FL-2IN. The molar
extinction coefficient becomes smaller with the increasing size of
the halogen atom. This is apparently associated with a decrease in
5
ꢁ1
ꢁ1
5
ꢁ1
ꢁ1
3
. Result and discussion
3.1. Synthesis
The synthetic routes for the two-photon photosensitizers are
the oscillator strength of the S
substitution of the heavier atom [37]. Meanwhile, the fluorescence
quantum yields ( ) follow this order: FL0 (0.53) > FL-2BrN
0 1
/S transition as a result of the
shown in Scheme 1. Compound FL0 was prepared by a two-fold
Heck reaction between the aryl bromide and 1,4-
2
F
f

C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
11
ꢀ
Scheme 1. Synthetic routes to chromophores FL0, FL-2BrN, FL-1IN, FL-2IN, and FL-2IS. (i) Pd
2
(dba)
3
, dppf, t-BuONa, toluene, 100 C; (ii) Pd(OAc)
2
, P(o-tolyl)
3
, Et
3
N, MeCN, reflux;
ꢀ
(
iii) t-BuONa, THF, 0e20 C.
(
0.37) > FL-1IN (0.27) > FL-2IS (0.25) > FL-2IN (0.22). These large
changes in are also attributed to a strong heavy atom effect. The
decreased fluorescence quantum yields indicate an increase in the
Ti:sapphire laser. The data were obtained by a two-photon excited
fluorescence method with fluorescein (80
M in water, pH ¼ 11) as
a reference [38]. TPA cross-section ( ) peak values of these five
chromophores in THF solution (2 ꢂ 10 M) were determined and
listed in Table 2. Fig. 2 shows the TPA spectra of FL0, FL-2IS, FL-1IN,
FL-2IN, and FL-2BrN with different halogen atoms. As shown in
Fig. 2, these chromophores have TPA bands centered at around
F
f
m
s
2
1
ꢁ6
ISC rate, therefore, an increased generation of
expected.
O
2
would be
3
.3. Two-photon absorption and emission
~
2
770 nm and the s peak values for compounds FL0, FL-2IS, FL-1IN,
FL-2IN, and FL-2BrN, are 2084, 2319, 2471, 2564 and 2707 GM, in
that order. In comparison with our previous reported ladder-type
oligo-p-phenylene based chromophores [23], these new chromo-
TPA spectra were determined by using laser pulses (<120 fs)
tunable from 730 to 860 nm generated by a mode locked
phores have larger TPA cross-section values, due to the extended
conjugation lengths. Similarly, the peak values of compounds
with longer -conjugation lengths (FL-2IS, FL-1IN, FL-2IN, and FL-
p-
1
.2
.0
1.2
1.0
.8
s
2
FL0
FL0
p
FL-2IS
FL-1IN
FL-2IN
FL-2BrN
FL-2IS
FL-1IN
FL-2IN
FL-2BrN
2BrN) are larger than that of compound FL0 with a shorter
p-
1
.8
.6
.4
.2
Table 1
Linear photophysical properties of compounds FL0, FL-2IS, FL-1IN, FL-2IN, and FL-
2BrN in THF.
.6
ꢁ1
ꢁ1
em (nm)c
v (cm )d
ꢁ1
e
Compounds
l
abs (nm)a
ε
max (M cm )b
l
D
F
f
.4
5
FL0
422
423
424
430
434
2.52 ꢂ 10
1.76 ꢂ 10
1.84 ꢂ 10
1.43 ꢂ 10
2.17 ꢂ 10
472
491
518
543
552
2510
3330
4279
4839
4925
0.53
0.25
0.27
0.22
0.37
5
5
5
5
FL-2IS
FL-1IN
FL-2IN
FL-2BrN
.2
0.0
0.0
a
Linear absorption peaks.
Molar extinction coefficient.
3
00
400
500
600
700
b
c
Wavelength/nm
One-photon excited emission peaks.
Stokes shift.
Fluorescence quantum yields determined by using Fluorescein (
d
e
Fig. 1. Normalized absorption (solid lines) and fluorescence (dash lines) spectra of FL0,
FL-2IS, FL-1IN, FL-2IN, and FL-2BrN in THF.
F
f
¼ 0.90 in
water, pH ¼ 11) as the reference.

12
C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
Table 2
Photophysical properties of compounds FL0, FL-2IS, FL-1IN, FL-2IN, and FL-2BrN.
(GM)a
s
0
(GM)b
F
D
c
Compounds
Maximum
s
2
2
FL0
2084
2319
2471
2564
2707
1105
580
667
564
1002
0.24
0.81
0.70
1.00
0.52
FL-2IS
FL-1IN
FL-2IN
FL-2BrN
a
Maximum TPA cross section value by the fluorescence method in THF.
b
c
0
TPA action cross section
s
2
¼
F
f
ꢂ
2
s .
Relative 1
O
2
yields that were normalized to that of FL-2IN.
conjugation length. It can be found that besides the effect of the
conjugation length on the value, the substituted halogen atoms
such as Br, I) at the phenyl ring of the core also affect the maximal
values. For example, the peak value of compound FL-1IN is
319 GM, while with the same -conjugation length, compound
FL-2BrN has a peak value of 2707 GM. This difference can be due
p-
s
2
(
s
2
s
2
2
p
s
2
to the introduction of the electron withdrawing halogen atoms in
the core, creating a donor-acceptor-donor (D-A-D) motif, leading to
the increased extent of charge transfer from the ends of the
molecule to the center, namely an increased intramolecular charge
transfer (ICT). As it has been known that large TPA is associated
with the extent of ICT for a given molecule. Obviously, compound
FL-2BrN has a stronger ICT, because of the stronger electron
withdrawing ability of bromine compared to that of iodine. Notably,
Fig. 3. Two-photon excited fluorescence (TPEF) spectra for the indenofluorene-based
chromophores in THF and their peak intensity versus the laser intensity at 800 nm
(inset).
As shown in Fig. 3, upon an excitation by 800 nm laser pulses, all
the chromophores emitted frequency-upconverted fluorescence
with emission maxima ( max) in the range of 498e573 nm. For
example, the two-photon excited emission peaks of FL0, FL-2IS, FL-
IN, FL-2IN, and FL-2BrN in THF were 498, 516, 527, 568, and
73 nm, in that order. Similar to their linear emission in THF,
l
although compound FL-2IS has a longer
p-conjugation length, its
1
5
s
2
value is smaller than those of compounds FL-1IN and FL-2IN,
which can be also attributed to the enhancement of ICT by incor-
porating the strong electron donating diarylamino groups into FL-
compound FL-2BrN has a red-shifted emission band compared to
that for compound FL0. From Figs. 1 and 3, one may find that the
TPE fluorescence spectra for compounds FL0, FL-2IS, FL-1IN, FL-
1
IN and FL-2IN. The result is also consistent with our previous
studies [24].
Two-photon excited fluorescence (TPEF) spectra of the two-
photon absorbing chromophores FL0, FL-2IS, FL-1IN, FL-2IN, and
2IN, and FL-2BrN in THF are basically the same as their linear
fluorescence spectra, which confirms that both emissions are from
the same excited state, although different excitation processes and
different selection rules are applied for TPEF and one-photon
excited fluorescence.
ꢁ
6
FL-2BrN were recorded in THF solution (2 ꢂ 10
M) at room
temperature by using an Ocean Optics s2000 spectrometer in
conjunction with a fiber coupler head (Fig. 3). Under these condi-
tions, the intensity for two-photon excitation was in an excitation
regime where the fluorescence signal showed a quadratic depen-
dence on the intensity of the excitation beam, as expected for two-
photon induced emission. As an example, the logelog plots show
slopes of 2.02 and 1.96 for compounds FL0 and FL-2IN, respectively
For the application of two-photon materials in fluorescence
0
imaging, it is important to know the TPA action cross section
s
2
0
(s
2
¼
s
2
ꢂ
F
f
), where
s
2
is the TPA cross section and
f
F is the
values for
fluorescence quantum yield, on condition that the
F
f
both one- and two-photon excited fluorescence are the same. As
listed in Table 2, all the five chromophores have much larger TPA
(
inset of Fig. 3).
action cross section values compared to most of commercial dyes.
0
The
s
2
values of FL0 and FL-2BrN (1105 and 1002 GM) are larger
than those of the others. FL-2IS, FL-1IN and FL-2IN show rela-
3500
3000
2500
2000
1500
1000
0
tively low
tively low
s
2
values of 580, 667, and 564 GM due to their rela-
values although they have large values. The two-
FL0
F
f
s
2
FL-2IS
FL-1IN
FL-2IN
FL-2BrN
photon excited fluorescence of these chromophores can be
directly observed with naked eyes (inset of Fig. 2) due to their high
active TPA action cross sections. It can be expected that these new
chromophores are good candidates for two-photon imaging
applications.
1
3
.4. Quantum yield of O
2
generation (
F
D
)
D
F is a very important measure of the efficiency of photosen-
1
sitization. A high O
2
generation efficiency is required for the PDT
1
application of photosensitizers.
O
2
generation efficiencies of
compounds FL0, FL-2BrN, FL-1IN, FL-2IS, and FL-2IN in air-
saturated toluene solution were evaluated by monitoring the
characteristic emission peak of singlet oxygen at ~1270 nm at room
temperature using 9,10-dibromoanthracene (DBA) as a reference
720
760
800
840
Wavelength/nm
(
F
D
¼ 0.85) [39,40]. The concentrations of all solutions were
Fig. 2. TPA spectra for the indenofluorene-based chromophores in THF measured by
the two-photon excited fluorescence method.
adjusted to give an optical density (OD) of 0.5 at 430 nm. Then, the

C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
13
1O
can be calculated according to the following equation:
2
D
generation yield (F ) of a sample under one-photon excitation
FL0
.
.
.
20
15
10
FL-2BrN
FL-1IN
FL-2IS
FL-2IN
Only DPBF
I
r
s
A
A
r
t
r
s
s
f
¼ f $ $
$
(1)
D
D
I
r
s
t
Where I is the emission intensity of singlet oxygen at ~1270 nm, A is
the absorbance of the solution, is the lifetime of singlet oxygen
t
phosphorescence in the selected solvent [41].
Under an excitation at 430 nm, five compounds all displayed the
1
characteristic O
2
O
emission band at ~1270 nm (Fig. 4), which con-
generated from the interaction between the
firms that the 1
.05
.00
2
photosensitizers and the molecular oxygen. The luminescence in-
tensities for chromophores FL0, FL-2BrN, FL-1IN, FL-2IS, FL-2IN
0
and DBA follow this order: 463, 1006, 1341, 1574, 1898, and 2305.
1
0
5
10
15
20
25
30
Obviously, the O
2
luminescence intensity of FL-2IN is significantly
Irradiation Time/min
stronger than those of the other photosensitizers with the same
1
OD, which indicates that FL-2IN has a higher O
ciency. According to Equation (1), the values of FL0, FL-2BrN, FL-
IN, FL-2IS, and FL-2IN were 0.17, 0.37, 0.49, 0.58 and 0.70, in that
order (Table 2). The values become larger with the increasing
size of the halogen atom. Compared with FL0, the O
2
generation effi-
Fig. 5. Plots of change in the absorbance of DPBF at 418 nm versus interval irradiation
time in the presence of the indenofluorene-based chromophores in air-saturated
toluene solution under a laser irradiation at 808 nm. A
F
D
1
0 t
is the initial absorption, A
is the absorption after irradiating for an interval of time.
F
D
1
2
generation
yields of FL-2BrN, to FL-1IN, to FL-2IS, and to FL-2IN, are enhanced
presence of the five photosensitizers were observed and the photo-
oxidation rates of DPBF for these compounds were in this order:
by factors of 2.2, 2.9, 3.4, and 4.1, in that order. The enhanced
1
generation of O
2
for the molecules with halogen atoms (Br, or I)
FL0 < FL-2BrN < FL-1IN < FL-2IS < FL-2IN. The results are in
incorporated is attributed to the increase of ISC rate induced by the
heavy atom effect. Correspondingly, decreased fluorescence quan-
tum yields were found for the molecules with halogen atoms in
1
agreement with their
2
O generation capability induced by one-
photon excitation. Similarly, the photo-oxidation rate of DPBF in
the presence of FL-2IN is obviously faster than those of the others
agreement with their increased ISC rates.
under two-photon excitation, also indicating that FL-2IN has a
1
Two-photon induced O
2
generation of the chromophores in air-
1
higher
2
O generation capability and can act as a better photo-
saturated toluene was also evaluated by a chemical trapping
method using 1,3-diphenylisobenzofuran (DPBF), a well-known
sensitizer for two-photon PDT.
singlet oxygen indicator [42]. In this method, DPBF reacts with
O to form peroxide, which leads to a decrease in the absorption of
2
1
3.5. Cytotoxicity against cancer cells
DPBF at 418 nm. The UV absorption at 418 nm was monitored
during the course of irradiation by a laser beam at 808 nm in the
presence of photosensitizers. The blank control experiments were
performed in the presence of only DPBF at the same conditions. The
curve of decrease in the absorption at 418 nm of DPBF was plotted
as a function of the irradiation time. As shown in Fig. 5, throughout
the irradiation time, no obvious changes of the absorption intensity
of DPBF were observed for the blank sample. However, significant
decreases of the absorption intensity of DPBF at 418 nm in the
Good biocompatibility and low dark cytotoxicity are essential
for photosensitizers in biological applications. To evaluate FL-2IN
as an effective photosensitizer for two-photon PDT of tumors, the
in vitro cytotoxicity against MCF-7 (human breast adenocarcinoma
cell line) cells was investigated by a MTT assay. The toxicities of
FL-2IN at varying concentrations were tested in the dark or under
a laser beam irradiation. As seen from Fig. 6a, after incubation for
24 h in dark, FL-2IN showed no obvious cytotoxicity effect on
MCF-7 cells in the concentrations range from 5 to 10 M. However,
m
the dark cytotoxicity increased with the increasing concentration
of compound FL-2IN. For example, when the concentration of
2
2
1
1
500
000
500
000
compound FL-2IN increased from 5
m
M, to 10
M, the viabilities of cells decreased from 96.4%, to 92.3%,
6.2%, and 78.2%, respectively. The photo-toxicity of FL-2IN was
mM, 20 mM, and
LF0
40 m
LF-2BrN
LF-1IN
LF-2IS
LF-2IN
DBA
8
conducted using a laser at 808 nm with a light intensity of
ꢁ
2
2
~
3.0 W cm . The laser beam was unfocused, and has a beam size
of ~4.0 cm . The irradiation time was 10, 20, or 40 min, giving a
ꢁ
2
ꢁ2
ꢁ2
light dose of 1800 J cm , 3600 J cm , and 7200 J cm , respec-
tively. The plate without compound Fl-2IN was used as a control.
As shown in Fig. 6b, laser irradiation alone caused almost no
cytotoxicity (<2% cell death) at all in the absence of Fl-2IN. In
contrast, in the presence of compound Fl-2IN in concentration
5
00
range from 0 to 40
sively with the increasing laser irradiation time. For example, at
the concentration of 40 M, the viabilities of cells are 71.5%, 56.7%,
mM, the viabilities of cells decreased progres-
0
m
1200
1250
Wavelength/nm
1300
1350
and 48.3% after irradiation for 10, 20, and 40 min, respectively.
Meanwhile, under the same irradiation time, increased cell death
was found with the increasing amount of drug loaded. For
example, after an irradiation of 40 min, the viabilities of cells are
84.8%, 76.6%, 64.2%, and 48.3% with increasing concentration of
Fig. 4. Luminescence of 1
and DBA, with an excitation at 430 nm in toluene solutions. Gaussian fits are shown for
every emission curve (solid line).
2
O sensitized with the indenofluorene-based chromophores,

14
C. Tang et al. / Dyes and Pigments 117 (2015) 7e15
Fig. 7. The TEM image of FL-2IN nanoparticles obtained by the reprecipitation method.
4. Conclusions
A series of indenofluorene-based chromophores have been
designed and synthesized as two-photon active photosensitizers
for PDT. Heavy atoms (Br, I) were incorporated into the backbone of
the indenofluorene-based chromophores to enhance their singlet
oxygen generation efficiencies. The TPA spectra for these chromo-
phores were determined by using the two-photon excited fluo-
rescence method. With the incorporated heavy atoms,
chromophores FL-2IS, FL-1IN, FL-2IN, and FL-2BrN exhibited
larger TPA cross section peak values compared to the chromophore
(FL0) without a heavy atom. The singlet oxygen generation effi-
ciencies of these photosensitizers under one- and two-photon
excitation have also been evaluated. FL-2IN showed a 410%
higher
atom effect. Moreover, the high TPA cross-section value of FL-2IN in
combination with its high value makes it a promising photo-
D
F value in comparison with FL0, due to the strong heavy
Fig. 6. (a) The dark cytotoxicity of FL-2IN at different concentrations towards MCF-7
cells. (b) Photo-toxicity of FL-2IN at different concentrations towards MCF-7 cells after
the 808 nm laser irradiation with different irradiation times. The control experiment
without any photosensitizers was performed under the same experimental conditions.
F
D
sensitizer for two-photon PDT. FL-2IN provided a moderate two-
photon PDT efficacy against cancer cells. These results suggest
that the introduction of heavy atoms into two-photon absorbing
chromophores can be an efficient strategy to enhance the two-
photon excited singlet oxygen generation efficiencies of photo-
sensitizers. Future work will focus on covalently coupling the two-
photon photosensitizers with targeting biomolecules to improve
their biocompativity and selectivity towards cancer cells in PDT.
FL-2IN from 5 to 40
mM. Notably, increased concentration of
compound FL-2IN also leads to its increased dark cytotoxicity.
Considering the dark cytotoxicity induced by compound FL-2IN,
one may still observe an increase of cell death induced by the
increased 1
2
O generation. For example, after an irradiation for
4
2
0 min, the death rate of MCF-7 cells increases from 11.6% to
9.9%, when the concentration of compound FL-2IN increases
M. These preliminary results indicate the feasibility
from 5 to 40
m
Acknowledgments
of employing two-photon photosensitizer for PDT and FL-2IN can
act as an efficient photosensitizer for two-photon photodynamic
treatment on cancer cells. It should be noted that FL-2IN can be
dispersed in water to form nanoparticles using a reprecipitation
method with the aid of DMSO, and the size of the resulting
nanoparticles is about 10 nm. (Fig. 7) [43]. The linear absorption
and emission spectra for the FL-2IN nanoparticles in aqueous so-
lution are basically the same as those of FL-2IN in THF except for
slightly red-shifted absorption and emission bands for the latter.
However, the fluorescence quantum yield for FL-2IN in aqueous
solution decreases to 0.17 due to a stronger solvent-solute inter-
This work was in part supported by the Natural Science Foun-
dation of China (Nos 21102144 61325026, and 11104266), in part
supported by 100 Talents Programme of the Chinese Academy of
Sciences, in part supported by Special Project of National Major
Scientific Equipment Development of China (2012YQ120060) and
The CAS/SAFEA International Partnership Program for Creative
Research Teams.
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