Journal of Innovative Optical Health Sciences

Vol. 8, No. 1 (2015) 1540001 (8 pages)

.c The Authors
#
DOI: 10.1142/S1793545815400015

Studies on photodynamic mechanism of a novel chlorine

derivative (TDPC) and its antitumor e®ect for
photodynamic therapy in vitro and in vivo

Ying Ye*, Lai-Xing Wang†, Dan-Ping Zhang*, Yi-Jia Yan*

and Zhi-Long Chen*,‡
*Department of Pharmaceutical Science & Technology
College of Chemistry and Biology, Donghua University
Shanghai 201620, P. R. China
†
Changhai Hospital, Second Military Medical University
Shanghai 200433, P. R. China
‡
[email protected]

Received 31 October 2013

Accepted 13 December 2013
Published 19 February 2014

Photodynamic therapy (PDT) represents a promising method for treatment of cancerous tumors.
The chemical and physical properties of used photosensitizer (PS) play key roles in the treatment
e±cacy. In this study, a novel PS, 5,10,15,20-tetrakis((5-dipropylamino)pentyl)-chlorin (TDPC)
which displayed a characteristic long wavelength absorption peak at 650 nm were synthesized. It
also shows a singlet oxygen generation rate of 4.257 min
1 . Generally, TDPC is localized in
mitochondria and nucleus of cell. After light irradiation with 650 nm laser, it can kill many types
of cell, in addition, TDPC–PDT can destroy ECA-109 tumor in nude mice and a necrotic scab
was formed eventually. The expression levels of many genes which regulated cell growth and
apoptosis were determined by RT-PCR following TDPC–PDT. The results showed that it either
increased or decreased, among which, the expression level of TNFSF13, a member of tumor
necrosis factor superfamily, increased signi¯cantly. In general, TDPC is an e®ective antitumor PS
in vitro and in vivo and is worthy of further study as a new drug candidate. TNFSF13 will be an
important molecular target for the discovery of new PSs.

Keywords: Photosensitizer; photodynamic therapy; tumor; chlorin; TDPC.

1. Introduction cancerous tumors.1,2 It takes e®ect through localiz-

Photodynamic therapy (PDT) has emerged as a ation of photosensitizer (PS) in the target tissues
promising new approach for treating and curing prior to radiation with an appropriate wavelength.3,4

This is an Open Access article published by World Scienti¯c Publishing Company. It is distributed under the terms of the Creative
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cited.

1540001-1
Y. Ye et al.

PDT has potential advantage over surgery and 2. Material and Methods
radiotherapy due to its tissue-sparing properties.
2.1. Materials
However, most of PSs also display only a slight pre-
ference for malignant cells, resulting in signi¯cant TDPC was synthesized in our laboratory and
skin photosensitivity and high uptake by healthy Patent has been applied. All other chemicals and
cells and tissues. To enhance the treatment of cancer, reagents were of analytical grade and used without
new PS with absorption spectra at long wavelengths any puri¯cation.
(650–800 nm) which can improve tumor selectivity
and a quicker clearance are urgently needed to be
developed.5 2.2. Absorption and emission spectra
Chlorin-based PSs have been found to have ap- UV-Vis absorption spectrum was recorded on an
plications as phototoxic drugs for PDT.6–12 They ultraviolet visible spectrophotometer (Model V-530,
are often selected because of their characteristic Japan). Fluorescence spectra were measured on a
absorption spectra with a Soret band at approxi- Fluorescence Spectrometer (FluoroMax-4, France).
mately 415 nm and a usually narrow but very strong Slits were kept narrow to 1 nm in excitation and 1 or
Q-band around 650 nm.13 Such an inherent long 2 nm in emission. Right angle detection was used.
wavelength characteristic provides more e±cient All the measurements were carried out at room
light penetration in tumor tissues as compared to temperature in quartz cuvettes with path length of
those PSs absorbing light at short wavelength.14,15 1 cm. TDPC was dissolved in N, N-dimethylforma-
Therefore, e®orts in our laboratory were directed mide (DMF) as 5
M.
toward synthesis of chemically pure new longer
wavelength absorbing PSs related to it.
In the present study, a novel chlorin-based 2.3. Singlet oxygen generation
compound 5,10,15,20-tetrakis[(5-dipropylamino) 1,3-diphenylisobenzofuran (DPBF) was used as a 1O2
pentyl]-chlorin (TDPC) (see Fig. 1) was studied trapping reagent in DMF solution. DPBF and TDPC
and its photophysical and photochemical proper- were dissolved in DMF. Two groups were placed in
ties have been evaluated. The localization pattern sealed quartz cuvettes and tested in each experiment:
of TDPC was determined. We further explored (1) DPBF and TDPC (5  10
5 M, 5  10
7 M); (2)
the changes in gene expression that occurred in DPBF(5  10
5 M). They were all at a reagent grade
response to TDPC–PDT. The photosensitizing and used without further puri¯cation. A 5 mW Nd:
antitumor activities in vitro and in vivo were YAG laser (650 nm) was used as the light source. The
evaluated. absorbance of the solution at 410 nm was measured
every 10 s for a 120 s period with an UV-Vis spec-
trophotometer. Ratio of Singlet oxygen was carried
out according to the equation mentioned below: In
A0 =A ¼ kt, where A0 is absorbance in t0 ; A is
N
absorbance in t; k is the reaction rate constant[s
1 ]
and t is the time [s].16

N 2.4. In vitro photosensitizing e±cacy

NH N
2.4.1. Cell lines and cell culture
N HN Human esophageal carcinoma Eca-109 cell line was
N
purchased from Chinese Academy of Sciences. The
cells were cultured in RPMI 1640 medium, sup-
plemented with 10% fetal calf serum and incubated at
37  in a humidi¯ed atmosphere containing 5% CO2.
N
Cell viability was detected via 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
Fig. 1. Chemical structure of TDPC. and expressed as the percentage of control cells.

1540001-2
 Studies on photodynamic mechanism of novel chlorine derivative (TDPC)

All cells were incubated at 37  in 5% CO2 in a hu- 2.5. In vivo photosensitizing e±cacy
midi¯ed incubator.
A total of 18 nude BALB/C mice (9 males and 9
females, weighing 15–20 g) were used for the study.
2.4.2. Phototoxicity studies The mouse tumor models were set up by subcu-
taneous injection of 1  10 6 Eca-109 cells in the °ank.
The MTT assay was used to monitor the cytotox- Experiments were initiated when tumors reached
icity mediated by TDPC–PDT. Brie°y, Eca-109 1 cm in diameter (between 21 and 28 days after tumor
cells, which were incubated in media containing 10, injection). The tumor volume ðTVÞ ¼ 1=2  length
1, 0.1, 0.01, 0.001
M TDPC for 24 h, were irra- width: 2 The mice were randomly divided into three
diated at 650 nm by a Nd:YAG laser and the MTT groups: laser radiation group (6 mice), treatment
assay was then used to analyze cellular sensitivity. group (6 mice) receiving 8
M TDPC without laser
All cells were seeded at 2000 cells per well in 96- application, and TDPCþ laser radiation group
well plates, each group in triplicate at least, and (6 mice) which was given 8
M TDPC followed by
then incubated in a cell culture incubator with 5% laser radiation after 24 h. The mice were restrained in
CO2 at 37  C. Five microliter of MTT (5 mg/mL) rat holders and exposed to laser ( ¼ 650 nm) with a
reagent (Sigma, MO, USA) was added to each well light dose of 78 J/cm2. Tumor regression was eval-
and incubated for 4 h at 37  C. At the end of the uated every 5 days for 20 days.
incubation period, the medium was removed and
the formazan complex was solubilized with 100
L
DMSO. Absorbance of the complex was measured 2.6. Statistical analysis
with a micro-plate reader (Bio-Rad, California, One-way ANOVA and the Student's t-test were
USA) at a wavelength of 570 nm.17 used to measure di®erences using the SPSS16.0
software package. Data were reported as mean 
SD and p values <0.05 were considered signi¯cant.
2.4.3. Intracellular localization
Eca-109 cells were grown in 8-wells plates on poly-L-
lysine coated coverslips, incubated in the dark at 3. Results
37  C with 4
M TDPC for 24 h, then rinsed in the
3.1. UV-Vis absorption spectrum
medium and incubated with 0 nm MitoTracker
Green (Invitrogen, Carlsbad, CA) for 30 min at 37  C. The UV-Vis absorption spectrum of TDPC (see
After washing with PBS, cells were re-incubated Fig. 1) was determined in DMF at 0.5
M. As can
with 1 mg/mL Hoechst 33258 (Beyotime, Nanjing, be seen, the TDPC had an elevated absorption peak
China) for 10 min at 37  C. Double-stained cells at 650 nm (see Fig. 2), suggesting that TDPC might
were imaged under a confocal microscope (LSM 510 be an e®ective PS.
META, Carl Zeiss, G€ ottingen, Germany). Images
were analyzed with the LSM Image Browser 0.16
Version 2.8. TDPP
0.14

0.12
2.4.4. Real-time PCR 0.10
Total RNA was extracted from cells using Trizol
Abs

0.08
reagent (Invitrogen, Carlsbad, CA) according to the
0.06
manufacturer's protocol. cDNAs were synthesized
0.04 650nm
using M-MLV reverse transcriptase (Promega) for
real-time PCR analysis. Real-time PCR was per- 0.02
formed using SYBR Master Mixture (TAKARA, 0.00
Japan) to measure mRNA levels of selected genes.
300 400 500 600 700 800
Data were analyzed using the standard curve anal- Wavelength(nm)
ysis method with Light-Cycler 480 Software (Roche
Applied Science). Fig. 2. UV-Vis absorption spectrum of TDPC in DMF.

1540001-3
Y. Ye et al.

3.2. Fluorescence spectrum 3.4. In vitro studies

Fluorescence spectra were measured on a Fluor- 3.4.1. Phototoxicity studies
escence Spectrometer. TDPC can be excited at Eca-109 cells were incubated with TDPC for 24 h
418 nm, and its emission was monitored at wave- and exposed to light as described above. After PDT,
lengths 651 and 722 nm (see Fig. 3). cells were incubated in the dark for 8 h. Then cell
viability was measured by MTT assay. MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
3.3. Singlet oxygen generation bromide, a yellow tetrazole), was reduced to purple
Diphenylisobenzofurane (DPBF) as an e®ective formazan in living cells. DMSO was added to dis-
singlet oxygen quencher has been widely used in solve the insoluble purple formazan product into a
many areas. Its conjugated structure is destroyed colored solution. The absorbance of this colored
after photo-oxidation reaction with singlet oxygen solution could be quanti¯ed by measuring at a
which can be measured by UV-Vis spectropho- certain wavelength by a spectrophotometer. The
tometer. Solution of DPBF and TDPC has a strong results were compiled by origin system. As shown
absorption peak at 413 nm. Monitoring the changes in Fig. 5. It could be seen that at examined con-
in intensity of the Q band can prove singlet oxygen centrations no apparent cytotoxic e®ects of the
production generation. As shown in Fig. 4, sin- drugs were observed without light. The survival
glet oxygen generation rates of TDPC in DMF is fraction still reached 75.20  0.093% when concen-
4.257 min
1 . tration of drug was 10
M. In the light/no drug

2500000
Relative flourescence intensity(a.u.)
Relative fluorescence intensity(a.u.)

2500000
418nm
2000000 651nm
2000000

1500000 1500000

1000000 1000000

500000 500000
520nm547nm 597nm 722nm
0 0
350 400 450 500 550 600 650 600 650 700 750
Excitation wavelength(nm) Emission wavelength(nm)

(a) (b)

Fig. 3. Excitation and emission spectrum of TDPC in DMF. (a) Excitation spectrum of TDPC; (b) Emission spectrum of TDPC,
which was excited at 418 nm, and the peak was at 651 and 722 nm.

0.5
0min
0.4
2min
4min 0.4
6min
0.3 8min
0.3
In A 0/A r

10min
Abs

0.2
0.2

0.1 0.1

0.0 0.0
300 350 400 450 500 550 600 0 2 4 6 8 10
Wavelength(nm) Irradiation time(min)

(a) (b)

Fig. 4. (a) Absorption spectra for the photo-oxidation of DPBF photosensitized by TDPC; (b) First-order plots for the photo-
oxidation of DPBF photosensitized by TDPC(k ¼ 4:257 min
1 Þ.

1540001-4
 Studies on photodynamic mechanism of novel chlorine derivative (TDPC)

2
0 J/cm
120 2
5 J/cm
2
10 J/cm
2
100 20 J/cm
2
40 J/cm

Survival(% of control)
80

60

40

20

0
-3 -2 -1 0 1
TDPP Concentration(IgM)

Fig. 5. Cytotoxicity to Eca-109 cells with di®erent concentrations of TDPC and di®erent laser dose.

Fig. 6. The intracellular localization of TDPC in Eca-109 cells with MitoTracker Green and Hoechst 33258, respectively.

groups, decreased cell viability was not noticed 3.4.3. The changes of gene expression in
(p > 0:05). Survival of cells dropped only by a ECA-109 cells treated by TDPC–PDT
combination of the PS and light. Following PDT,
when the concentration of TDPC was increased, the To explore the changes of gene expression after
viability of cells was declined. However, the PDT TDPC–PDT, total cellular RNA was harvested
e®ect was not strong at 0.01 and 0.001
M concen-
tration but opposite at the concentration above
0.1
M. Besides that, there was no signi¯cant dif-
ference between light doses. The viability of cells
appeared gradient relation only at 0.1
M concen-
tration of TDPC. It was indicated that TDPC may
be an excellent PS and pro-oxidant in biological
applications.

3.4.2. Subcellular localization of TDPC

Photodynamic e±cacy is principally determined by
the subcellular localization of a PS. The localiz-
ation of TDPC (max ¼ 650 nm) in cancer cells was
analyzed by MitoTracker Green and Hoechst 33258
Fig. 7. The Changes of gene expression in Eca-109 cells after
counterstain. It was found that TDPC initially TDPC–PDT. RT-PCR results showed the expression changes
localized in mitochondria with some accumulation of 8 genes in Eca-109 cells treated by PDT with 2 or 4
M
in the nucleus in Eca-109 cells (see Fig. 6). TDPC, or with 4
M TDPC with no photo excitation after 24 h.

1540001-5
Y. Ye et al.

Fig. 8. In vivo photosensitizing e±cacy of TDPC against lung cancer in nude mice bearing ECA-109 tumors (6 mice/group). The
mice in PDT group were treated with laser light (698 nm, 78 J/cm 2 Þ at 24 h post injection of the drug. The mice in PS group were
treated with 8
M TDPC, but not exposed to light. The control mice were not subjected to any PS or light.

from ECA-109 cells after 24 h incubation in medium days after treatment. The normal skin phototoxicity
containing 2 or 4
M TDPC followed by irradiation subsided within 4 days. In contrast, the tumors in PS
with a dose of 0.18 J/cm2 light ( ¼ 650 nm). The or control group continued to grow and were sig-
expression levels of genes involved in apoptosis and ni¯cantly larger than in PDT-treated group after 10
cell growth were then detected by real-time PCR days post-treatment (see Fig. 8).
assay (see Fig. 7).
During TDPC–PDT, some genes involved in
apoptosis such as Bcl-2 and X-linked inhibitor of 4. Discussion
apoptosis (XIAP) were signi¯cantly up-regulated, An ideal PS should have absorption spectra at long
and the tumor necrosis factor superfamily member wavelengths, which allows deeper tissue penetration
13 (TNFSF13) also greatly increased after PDT and decreases nonspeci¯c lesions.18 For instance,
with the higher dose of TDPC (4
M). TDPC– 630 nm light penetrates less than 0.5 cm whereas
PDT at the dose of 2
M caused hypoxia-inducible 700 nm light reaches a depth of nearly 0.8 cm.19
factor-1a (HIF-1a) to be down-regulated, however TDPC can be excited by 650 nm light. Both in vitro
increasing the dose of TDPC furthermore could and in vivo data supported that TDPC might be an
promote HIF-1a expression. ideal PS.
Not only many PSs are bright °uorophores, but
also they tend to emit in the nearinfrared (NIR)
3.5. The e±cacy of TDPC–PDT against
portion of the spectra that is useful for in vivo
esophagus cancer in vivo imaging. Excited by 418 nm light, TDPC can emit
To determine the e®ects of TDPC–PDT on esopha- red °uorescence at 651 and 722 nm, which means
gus cancer in vivo, 1  10 6 ECA-109 cells were that it has the prospects to become a new reagent for
injected into the axilla of nude mice to form solid targeting °uorescence imaging in tumor diagnosis.
tumors (1 cm3 in size). 100
L of 8
M TDPC was In numerous PDT studies, it has been demon-
then injected into the tail vein and 24 h later the strated that generation of 1O2 is responsible for the
tumor was irradiated with 130 mW/cm laser light for initiation of cell death.20–24 The singlet oxygen was
10 min (78 J/cm2,  ¼ 650 nm). As shown in Fig. 8, identi¯ed as ROS involved in cell photosensitization
PDT-treated tumors began to shrink within ¯ve days by TDPC.
and became dark, hardened and dried over the course TDPC showed signi¯cant concentration and
of the next 10 days, eventually formed a scab by 20 light dose dependence and induced no dark toxicity

1540001-6
 Studies on photodynamic mechanism of novel chlorine derivative (TDPC)

in the range of concentrations used in the present 81101298), Foundation of Shanghai govern-
photodynamic studies via MTT assay. ment (13431900700, 13430722300, 13ZR1441000,
The chemical properties of a PS determine its 13ZR1440900), Foundation of Donghua University
subcellular localization, which in turn a®ect the (No. 11D10501; 12D10515) and Foundation of Yiwu
cytotoxicity of photoactivation. Some PSs show a Science and Technology Bureau (2011-G1-15; 2012-
broad distribution, while some may localize more G3-02).
speci¯cally. In general, the PSs that localize in
mitochondria show more e®ectiveness. We found
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1540001-8