Highly efficient organic photosensitizer with aggregation-induced emission for imaging-guided photodynamic ablation of cancer cells

Article abstract of DOI:10.1016/j.tetlet.2018.05.088

We design an organic photosensitizer with a donor-π-acceptor configuration. The photosensitizer exhibits aggregation-induced emission characteristics and efficient singlet oxygen production in the aggregated state. It is then enveloped into the water-soluble micelle to afford a nanoprobe. The water-soluble nanoprobe keeps the photosensitizer in the aggregation state and is used for imaging-guided photodynamic ablation of cancer cells.

Full text of DOI:10.1016/j.tetlet.2018.05.088

Accepted Manuscript
Highly efficient organic photosensitizer with aggregation-induced emission for
imaging-guided photodynamic ablation of cancer cells
Yipeng Li, Qi Yu, Pengli Gao, Huiran Yang, Tianci Huang, Shujuan Liu, Qiang
Zhao
PII:
S0040-4039(18)30726-3
https://doi.org/10.1016/j.tetlet.2018.05.088
TETL 50035
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
28 March 2018
18 May 2018
30 May 2018
Please cite this article as: Li, Y., Yu, Q., Gao, P., Yang, H., Huang, T., Liu, S., Zhao, Q., Highly efficient organic
photosensitizer with aggregation-induced emission for imaging-guided photodynamic ablation of cancer cells,
Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.05.088
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Graphical Abstract
Highly efficient organic photosensitizer with
aggregation-induced emission for imaging-
guided photodynamic ablation of cancer cells
Yipeng Li†, Qi Yu†, Pengli Gao, Huiran Yang, Tianci Huang,
Shujuan Liu, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors,
Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing,
2
10023, P. R. China.
We design an organic photosensitizer with a donor--acceptor configuration. The photosensitizer exhibits
aggregation-induced emission characteristics and efficient singlet oxygen production in the aggregated state. It
is then enveloped into the water-soluble micelle to afford a nanoprobe. The water-soluble nanoprobe keeps the
photosensitizer in the aggregation state and is used for imaging-guided photodynamic ablation of cancer cells.
A nanoprobe enveloped with aggregation-induced emission-active organic photosensitizer has been designed and
synthesized for imaging-guided photodynamic ablation of cancer cells.

1
Tetrahedron Letters
Highly efficient organic photosensitizer with aggregation-induced emission for
imaging-guided photodynamic ablation of cancer cells
Yipeng Li†, Qi Yu†, Pengli Gao, Huiran Yang, Tianci Huang, Shujuan Liu, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing
University of Posts and Telecommunications (NUPT), Nanjing, 210023, P. R. China.
Corresponding E-mail: iamqzhao@njupt.edu.cn (Q. Zhao). †Y. Li and Q. Yu contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We design an organic photosensitizer with a donor--acceptor configuration. The
photosensitizer exhibits aggregation-induced emission characteristics and efficient singlet
oxygen production in the aggregated state. It is then enveloped into the water-soluble micelle to
afford a nanoprobe. The water-soluble nanoprobe keeps the photosensitizer in the aggregation
state and is used for imaging-guided photodynamic ablation of cancer cells.
Available online
2
009 Elsevier Ltd. All rights reserved.
Keywords:
Photodynamic therapy
Photosensitizer
Donor--acceptor configuration
Aggregation induced emission
Introduction
atoms into the molecular structure is one of the most widely used
9
methods to improve the ISC efficiency, but these halogen atoms
Cancer remains one of the most commonly diagnosed diseases
and causes a high death rate. Up to now, the clinic treatments for
10
cause the increased dark toxicity. Additionally, since most
1
hydrophobic photosensitizers suffer from the aggregation in the
cancer mainly rely on surgery, chemical therapy and radiation
aqueous media through - stacking, the remarkable emission
2
therapy, which aim to remove the tumor tissues and reduce the
1
11
2
quenching and decreased production of O are observed.
recurrence rate in patients. These treatments have their merits and
demerits. Surgery is rendered as the most directly therapeutic
option to remove the tumor tissues, but the effectiveness is
usually dependent on the healthy condition of the individual
patient and the stage of the cancer, thus giving rise to the
unpredictable recurrence rate. The chemical therapy and radiation
therapy are also commonly used in the clinic applications, bu3t
they induce the unavoidable invasiveness and systemic toxicity.
In contrast, photodynamic therapy (PDT) has emerged as a novel
measurement for cancer therapy. It utilizes photosensitizers to
generate reactive oxygen species (ROS) to damage the tumorous
Fortunately, fluorophores with aggregation-induced emission
(
AIE) that were originally reported by Tang’s group have recently
12
utilized for biological applications. These fluorophores exhibit
weak emission when soluble in solution, while emit brightly in
the aggregated state. Some of these fluorophores have been
2
c
reported to show efficient ISC efficiency in the aggregation
13
state,
which paves the way for the development of
photosensitizers with AIE for PDT. For example,
tetraphenylethene derivatives are the most commonly employed
14
as scaffolds to build AIE-active photosensitizers.
In this work, we proposed to prepare an organic AIE-active
photosensitizer (1) with donor--acceptor (D--A) configuration.
We anticipate that the introduction of quinoline–malononitrile
moiety in 1 will give rise to AIE characteristics and the spatially
4
cells or tissues under light activation. Since the phototoxicity
towards cancer cells is only triggered under irradiation, the PDT
process exhibits specific spatiotemporal selectivity and minimal
invasiveness.
separated electron donor and acceptor moieties will result in the
Photosensitizers are involved in the PDT process as the
1
efficient
O
2
generation in the aggregation state. By
was easily
primary element that not only generates cytotoxic singlet oxygen
encapsulating into amphiphilic molecules,
1
1
(
2
O ) for cancer therapy but also can serve as an imaging agent to
enveloped into the water-soluble micelle to afford a nanoprobe.
The nanoprobe bearing 1 has been demonstrated to show bright
emission and efficient PDT efficiency. It can be used for imaging
and photodynamic ablation of cancer cells. The unique properties
of the nanoprobe warrant it as a novel photosensitizer for
imaging-guided cancer therapy.
label the distribution of the tumor. The reported photosensitizers
5
include
phosphorescent
transition-metal
complexes,
6
7
nanoparticles and organic compounds. Organic compounds
have attracted great research interest because of large molar
extinction coefficient and easy metabolism. However, compared
to transition-metal complexes, organic compounds have
8
relatively lower intersystem crossing (ISC) efficiency, resulting
Results and discussion
1
in the decreased generation of O
2
. The introduction of halogen
The synthesis route is shown in Scheme 1 by a six-step

2
Scheme 1. Synthetic route of 1.
Fig. 2 (a) The absorption spectra of ABDA in the presence of 1 in the mixture
15
procedure. 2 was prepared according to a reported method.
3
containing DMSO and water (1:200, v/v) under irradiation (white light, 100
was synthesized by the coupling of carbazole and 4-
bromobenzaldehyde through Ullmann reaction. The reaction of 2
with 3 in acetonitrile catalyzed by piperidine afforded 1. In 1, the
quinoline-malononitrile (QM) moiety (electron donor) and
carbazole (electron acceptor) were modified to obtain the
donor-acceptor system. The introduction of QM moiety aimed
at retaining the AIE characteristics. The intramolecular charge
transfer within 1 is feasible to lower the energy gap between the
-2
mW cm ) over different periods of time; (b) The decomposition rate of
0
ABDA by 1; A and A are the absorption of ABDA monitored at 400 nm in
the presence of 1 before and after irradiation, respectively.
2
+
mixture of 1 or Ru(bpy)
3
in the ABDA solution was irradiated
-2
with a white light lamp at 100 mW cm . The absorption band of
ABDA was observed to be gradually decreased in the presence of
photosensitizer under irradiation, indicating the degradation of
1
ABDA when O
2
was generated in solution. The decomposition
-3
-1
S
1
and T
1
states, which will facilitate the increased ISC efficiency,
rate constant of 1 was estimated to be 3.39  10 s (Fig. 2). The
O quantum yields of 1 was calculated to be 0.12. The capacity
of 1 to produce O agreed well with small ST value in the
1
1
resulting in the increasing efficiency of O
Supporting information).
2
generation (Fig. S1 in
2
1
6
1
2
The AIE characteristics of 1 was investigated by monitoring
the emission spectra in the mixture of dimethyl sulfoxide and
results of DFT calculation, suggesting the possibility to utilize 1
as a photosensitizer in intracellular environments.
The nanoprobe was then obtained by encapsulating the
hydrophobic organic photosensitizer 1 with amphiphilic DSPE-
mPEG5000 through a modified nanoprecipitation method (Fig.
water (DMSO/H
intensity was observed to be gradually enhanced along with an
increased fraction of water in DMSO/H O mixture. Interestingly,
2
O). As illustrated in Fig. 1a, the emission
2
when the fraction of water was increased, the bathochromic
effect was enhanced. The fraction of water reached to be 90%,
the emission spectrum of 1 was bathochromically shifted to far-
red region (em = 639 nm), which is advantageous to deep tissue
penetration and minimize noise from the autofluorescence in
biological applications. The absorption spectrum of 1 was
observed to localize in the white light (> 400 nm), which paves
the way for minimized photo-damage (Fig. 1b). In order to better
understand the optical properties of 1, the density functional
theory (DFT) calculations were carried out using the Gaussian 09
package at the B3LYP level. The HOMO and LUMO distribution
are respectively localized in carbazole and malononitrile (Fig. 1c),
which demonstrated intramolecular charge transfer within
molecular systems. The spatially separated electron donor and
acceptor moieties resulted in small ST value that was estimated
3
a). The nanoprobe prepared exhibited good monodispersity and
homogeneity with the average particle size of 48 nm (Fig. 3b).
The dynamic light scattering result (54.8 nm) (Fig. 3c) was in
agreement with that detected in transmission electron microscopy.
The absorption band of 1 at 438 nm was observed in the
nanoprobe solution (Fig. S2a in Supporting information), which
demonstrated that 1 was enveloped into the micelle. The mass of
1
doped in the nanoprobe solution (2 mL) was assessed to be 1.5
mg through calibration analysis based on UV/Vis absorption
spectra. The nanoprobe was highly stable and can be stored in
phosphate buffer saline (PBS, pH = 7.4). The nanoprobe
exhibited intense AIE-active emission at 600 nm (Fig. S2b in
Supporting information). The O
was calculated to be 0.20 (Fig. S3 in Supporting information),
which was ascribed to the relatively small integrated area of
absorption band. These results revealed that the optical properties
1
2
quantum yield of naonmicelle
to be 0.61 eV, which makes it possible to enhance ISC efficiency
1
for O
2
g1eneration.
The O
2
generation of 1 was assessed by using 9,10-
anthracenediyl-bis(methylene) dimalonic acid (ABDA), as an
2
+
indicator and Ru(bpy)
3
as the standard photosensitizer. The
a
b
3
2
1
00
00
00
1.0
fw (vol %)
0%
1
9
0.5
0
%
0
5
0.0
200
00
600
700
800
300
400
500
600
Wavelength (nm)
Wavelength (nm)
c
LUMO
HOMO
Fig. 3 (a) Schematic illustration of the formation of the nanoprobe through
nano-precipitation method; (b) TEM image of the nanoprobe; (c)
Hydrodynamic size distribution of the nanoprobe.
Fig. 1 (a) Photoluminescence spectra of 1 (8 M) in DMSO/water mixtures
with different fraction of water; (b) The absorption spectrum of 1 (8 M) in
DMSO; (c) Chemical structure and HOMO-LUMO distribution of 1.

3
b. + nanoprobe
c. + nanoprobe
Red channel
Bright field
Overlay
a. Controlgroup
.07%
11.5%
without irradiation
under irradiation
1
e6
1
e6
1
e5
e4
12.9%
42%
0
1e5
1e5
1e4
1e3
1
1
e4
e3
1
e3
00
88.8%
1
69.6%
11.9%
19.2%
37.5%
1
a
b
100
100
1
0
10
0
1
0
0
0
0
10 100 1e3 1e4 1e5
FITC
0 101001e3 1e4 1e5
FITC
0
101001e31e41e51e6
FITC
Fig. 6 Flow cytometry analysis of cell death induced by the nanoprobe-
mediated PDT.
generation of the nanoprobe in cells under irradiation. When the
nanoprobe incubated cells were treated with DCFH-DA for 5 min,
the luminescence from the emission channel (505  525 nm) was
detected after white light irradiation (100 mW cm ) for 5 min
Fig. S6c in Supporting information). We also conducted the
-2
(
similar experiments using blank cells or the nanoprobe loading
cells without irradiation (Fig. S6a-b in Supporting information).
The relatively weak luminescence was observed in these control
groups (Fig. S7 in Supporting information), which demonstrated
that the emission enhancement was attributed to the nanoprobe
induced photodamage.
Fig. 4 Confocal laser scanning microscopy images of HeLa cells incubated
-
1
without (a) and with (b) the nanoprobe (18 g mL ). The image of left
column collected the emission from 580  650 nm; Excitation wavelength
was 405 nm. The middle column is bright field image. The right column is
the overlay image.
of 1 were not affected when it was encapsulated into the
Furthermore, the anticancer effects of the nanoprobe were
investigated using HeLa cells as a model. The cell death induced
by the nanoprobe mediated PDT was studied using two
fluorescent indicators, Annexin V-FITC/propidium iodide(PI)
that enable to monitor and differentiate the cellular apoptotic
stages. As illustrated in Fig. 5, different from the blank cells and
the nanoprobe loading cells in dark, cells treated with the
nanoprobe followed by irradiation exhibited bright green
fluorescence from Annexin V-FITC in the cell membrane and red
fluorescence from PI in the cell nucleus. These results suggested
the good anticancer efficiency of the nanoprobe. Additionally, the
real-time imaging was conducted to monitor the long-term
cellular apoptosis process (Fig. S8 in Supporting information).
The cells showed green fluorescence after 1 h, which proved that
the early apoptosis occurred in cells. After 2.5 h, we observed the
cells death, resulting in the enhancement of red emission in
nucleus.
nanostructure. Additionally, the stable structure of nanoprobe
1
enhanced O
2
generation efficiency of 1 and facilitated the PDT
process in cells.
The cytotoxicity of nanoprobe is essential to be considered
when it was used for the biological applications in cells. We first
examined the influence of the nanoprobe on cell viability through
the 3-(4,5-dimethyl-w-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide (MTT) assay (Fig. S4 in Supporting information). The
-
1
nanoprobe with the concentration ranged from 0 to 400 g mL
was added to the incubation buffer. When the incubation
-1
concentration was increased to 400 g mL , the viability of
HeLa cells remained more than 90% after 24 h, indicating low
cytotoxicity of the nanoprobe. We further used HeLa cells as a
model to study the possibility of utilization of the nanoprobe as a
potential imaging agent via confocal laser scanning microscopy
-1
(
Fig. 4). When cells were treated with the nanoprobe (18 g mL )
for 1 h, intense fluorescence collected from the red channel (em
580  650 nm) was observed in the cytoplasm (Fig. 4b).
The quantitative evaluation of the anticancer effects was also
conducted using flow cytometry. As illustrated in Fig. 6, the
proportion of apoptotic cells was gradually increased when cells
were incubated with the nanoprobe and exposure to light
irradiation for 5 min. The late-stage apoptotic cells reached to
=
Compared to the blank cells, The nanoprobe incubated cells
showed intense fluorescence, excluding the autofluorescence
interference (Fig. 4a, Fig. S5 in Supporting information).
We used 2,7-dichlorifluorescein-diacetate (DCFH-DA) as an
42%, which were sharply increased compared to the control
indicator, which can convert to 2,7-dichlorodihydrofluorescein
1
groups. The results of MTT assay were in agreement with those
detected in flow cytometry (Fig. S9 in Supporting information).
A dose-dependent cytotoxicity was observed in HeLa cells under
irradiation. When the concentration of the nanoprobe incubated
(
DCFH) in cells and undergo oxidation with O
2
to produce
1
fluorescent 2,7-dichlorofluorescein (DCF), to monitor the O
2
Brightfield
FITC
PI
Overlay
-1
was kept at 18 g mL , the varying irradiation time also resulted
in different degree of cell death (Fig. S10 in Supporting
information). These results demonstrated the good therapeutic
efficiency of the nanoprobe.
a. Controlgroup
Conclusions
b. + nanoprobe
without irradiation
In summary, we prepared an organic AIE-active
photosensitizer 1. It owned spatially separated electron donor and
acceptor, leading to low energy gap between the S
1
and T
1
states
1
and thus increased
2
O quantum yields. To stabilize 1 in
c. + nanoprobe
under irradiation
aggregation state and improve the biocompatibility, we
enveloped 1 into a water-soluble nanoprobe and employed the
nanoprobe for PDT process in cells. Different from the traditional
photosensitizer, the nanoprobe overcame the aggregation-induced
quenching in practical biological applications, and showed strong
fluorescence emission, which rendered 1 as a potential imaging
agent. The efficient therapeutic effects detected from the
Fig. 5 Confocal microscopy images of annexin V-FITC- and PI-labeled HeLa
-
1
cells. (a) blank cells; (b) Cells were treated with the nanoprobe (18 g mL )
without irradiation (100 mW cm 5 min); (c) Cells were treated with the
-
2
-
1
-2
nanoprobe (18 g mL ) with irradiation (100 mW cm 5 min).

4
nanoprobe demonstrated the possibility of its applications in
15. Sun W, Fan J, Hu C, et al. Chem. Commun. 2013; 49: 3890-3892.
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402-6406.
1
imaging-guided cancer therapy. Additionally, the successful
employment of the organic molecule bearing D--A structures
opens up a novel strategy to design metal-free photosensitizers
with excellent PDT process.
(
6
Acknowledgments
Y. Li and Q. Yu contributed equally to this work. The authors
acknowledge the National Natural Science Foundation of China
(51473078 and 21671108), National Program for Support of
Top-Notch Young Professionals, Scientific and Technological
Innovation Teams of Colleges and Universities in Jiangsu
Province (TJ215006), and Priority Academic Program
Development of Jiangsu Higher Education Institutions
(YX03001) for financial support.
Supplementary data
Supplementary data (details of the organic photosensitizer
synthesis, NMR, cell culture) associated with this article can be
found in Supporting Information.
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5
Highlights

An AIE-active organic photosensitizer is
1
prepared and shows efficient O
2
generation
under lamp.


A nanoprobe envolped with the photosensitizer
has been prepared.
The nanoprobe is used for imaging-guided
photodynamic ablation of cancer cells.

6
A nanoprobe enveloped with aggregation-induced
emission-active organic photosensitizer has been
designed and synthesized for imaging-guided
photodynamic ablation of cancer cells.