Journal of Innovative Optical Health Sciences

Vol. 8, No. 1 (2015) 1540003 (11 pages)

.c The Authors
#
DOI: 10.1142/S1793545815400039

Anti-tumor activities of a novel chlorin derivative for

photodynamic therapy in vitro and in vivo

Li-Jun Zhang*, Lai-Xing Wang†, Wei-Li 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 30 October 2013

Accepted 5 January 2014
Published 20 February 2014

In this study, a novel photosensitizer meso-tetra (3-pyrrolidinomethyl-4-methoxyphenyl) chlorin

(TPMC) was reported. It displays a characteristic long wavelength absorption peak at 656 nm
and it shows a singlet oxygen quantum yield of 0.48. After light irradiation with 650 nm laser, it
can kill Eca-109 and SMMC-7721 cells in vitro (25 mW/cm2, 1.2 to 3.6 J/cm 2 Þ and destroy
Eca-109 tumor in nude mice (50 mW/cm2, 90 J/cm 2 Þ. It has the perspective to be developed as a
new anti-tumor drug in photodynamic therapy (PDT) photodiagnosis, and deserves further
investigation.

Keywords: TPMC; chlorin; tumor; photosensitizer; photodynamic therapy.

1. Introduction through direct cellular damage, vascular shutdown

Photodynamic therapy (PDT) is emerging as a and activation of an immune response against
targeted cells.7
promising method for the treatment of a variety of
Photosensitizers in PDT play a critical role.8,9
oncological, dermatological, cardiovascular and
Selection of an appropriate photosensitizer is of
ophthalmic diseases.1–6 PDT destroys target cells in paramount importance for PDT.10 Factors that
the presence of oxygen when light irradiates a may in°uence the selection include conjugation
photosensitizer, generating highly reactive singlet compatibility and yield, quencher compatibility,
oxygen and/or other reactive oxygen species (ROS) photosensitizer hydrophobicity, excitation pro¯le,
such as superoxide ion and hydroxyl radicals. ROS singlet oxygen quantum yield, °uorescence quan-
then attack biological targets, causing destruction tum yields and photosensitizer dark toxicity.3
This is an Open Access article published by World Scienti¯c Publishing Company. It is distributed under the terms of the Creative
Commons Attribution 3.0 (CC-BY) License. Further distribution of this work is permitted, provided the original work is properly
cited.

1540003-1
L.-J. Zhang et al.

Chlorin-based photosensitizers have been found 2.2. Absorption and emission spectra
to have applications as phototoxic drugs for
UV-Vis absorption spectrum was recorded on an
PDT.11–14 They are often selected because of their
ultraviolet visible spectrophotometer (Model V-530,
high singlet oxygen quantum yields and spectro-
Japan). Fluorescence spectra were measured on a
scopic properties15: Characteristic absorption spec-
Fluorescence Spectrometer (FluoroMax-4, France).
tra with a Soret band at approximately 415 nm and
Slits were kept narrow to 1 nm in excitation and 1 or
a usually narrow but very strong Q-band around
2 nm in emission. Right angle detection was used.
650 nm, with a molar absorption coe±cient in the
All the measurements were carried out at room
range of 105 M
1 cm
1 . Temopor¯n and meso-
temperature in quartz cuvettes with path length of
tetahydroxyphenylchlorin have been used for eso-
1 cm. TPMC was dissolved in N, N-dimethylfor-
phageal cancer, head and neck cancer, early gastric
mamide (DMF) to get 5
M solution.
cancer and gastrointestinal cancer super¯cial
portion.16–18
We herein report the photobiological studies of a 2.3. Singlet oxygen quantum yield
novel Chlorin-based compound meso-tetra (3-pyr- DPBF was used as a 1 O2 trapping reagent in DMF
rolidinomethyl-4-methoxyphenyl) chlorin (TPMC) solution. In a typical experiment, 2 mL DMF solution
(see Fig. 1) using human esophageal cancer cells containing 20
M 1,3-diphenylisobenzofuran (DPBF)
(Eca-109) and human hepatocellular carcinoma and 0.5
M TPMC was placed in a sealed quartz
cells (SMMC-7721) in vitro and in vivo. The pho- cuvette. A 5 mW Nd:YAG laser (650 nm) was used as
tophysical and photochemical properties have been the light source. The absorbance of the solution at
evaluated. Our results show the e±ciency of TPMC 410 nm was measured every 10 s for a 120 s period
for suitable PDT applications. To further elucidate with an ultraviolet visible spectrophotometer.19 The
the cellular mechanism of action, the intracellular decrease of the absorbance caused by photobleaching
localization was tested for the purpose of deter- of DPBF was measured and corrected in all exper-
mining the primary photodamage site. iments. The natural logarithm values of absorption of
DPBF at 410 nm were plotted against the irradiation
time and ¯t by a ¯rst-order linear least-squares model
2. Experimental Section to get the singlet oxygen generation rate of the pho-
tosensitized process.20 The 1O2 quantum yield of
2.1. Materials TPMC in DMF was calculated using Methylene blue
TPMC (see Fig. 1) was synthesized in our labora- as a standard.
tory and is being applied for patent. All other
chemicals and reagents were of analytical grade and
2.4. In vitro experiments
used without any puri¯cation.
2.4.1. Cell lines and culture conditions
Human esophageal cancer cell line (Eca-109) and
OCH 3 human hepatocellular carcinoma cell line (SMMC-
N
7721) were obtained from the Type Culture Collec-
tion of the Chinese Academy of Sciences. All cell
culture related reagents were purchased from
Shanghai Ming Rong Bio-Science Technology Co.,
NH N Ltd. Cell lines were maintained in normal RPMI-1640
H3CO OCH 3 culture medium with 10% fetal bovine serum (FBS)
N HN
N
and 100 IU/mL penicillin and 100
g/mL strepto-
N
mycin. Both cell lines were kept in a CO2 incubator
with 5% CO2 and 100% relative humidity at 37  C.

N 2.4.2. Cellular uptake

OCH 3
Eca-109 and SMMC-7721 cells (1  10 4 cells/well)
Fig. 1. Chemical structure of TPMC in DMF. were grown overnight at 37  C in 96-well cell culture

1540003-2
 Anti-tumor activities of a novel chlorin derivative for PDT

plates. A 5
M solution of TPMC in culture medium described above was performed after a further incu-
with 4% serum was then exposed to cells from 30 min bation of 24 h after irradiation and compared to the
to 24 h in the dark, respectively. The solution was values of control cells without light irradiation.
prepared by diluting the stock solution (10 mM) in
dimethyl sulfoxide (DMSO) with proper cell culture 2.4.5. Intracellular localization
medium to the desired ¯nal concentration (5
M).
The highest DMSO concentration did not exceed Eca-109 and SMMC-7721 cells grown on coverslips
0.3% v/v. After incubation, cells were washed three were incubated with the solution (5
M) of TPMC
times with PBS and solubilized in 100
L/well for 4 h at 37  C in the dark. Then after removing the
DMSO. Drug cellular uptake was measured by solution with PBS, cells were stained with °uorescent
determining the °uorescence emission of TPMC with dye (Hoechst 33342) for cell nucleus diluted in the
a Spectro°uorometer (FluoroMax-4, France) at culture medium without serum. After washing with
420 nm excitation and 656 nm emission wavelengths. PBS, coverslips were ¯xed for 10 min at
4  C with
Results were expressed as the emission relative to the 4% paraformaldehyde and cells were then examined
total number of cells. by °uorescence with a confocal microscopy (LSM
410, Zeiss, Germany). TPMC was excited at 420 nm
and its emission was monitored at wavelengths
2.4.3. Toxicity in the dark
656 nm, and Hoechst 33342 was excited at 350 nm
Eca-109 and SMMC-7721 cells (1  10 4 cells/well) and blue °uorescence was detected at 461 nm.
grown in 96-well cell culture plates were incubated
for 24 h with concentration from 2 to 10
M of
TPMC. The solutions were prepared as described 2.5. In vivo experiments
above. Maximum DMSO concentration in the cell 2.5.1. Animal models
culture medium was < 0.3% v/v. Cell toxicity anal-
TPMC was evaluated in nude mice (10 mice/group)
ysis was performed at the end of the incubation time.
bearing Eca-109 tumors, implanted subcutaneously,
Brie°y, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-
and the tumors were allowed to grow to an ap-
trazolium bromide (MTT) dissolved in phosphate
proximate diameter of 5–7 mm.
bu®ered saline (PBS) (5 mg/mL) was added (10
L
per 100
L of medium) to all wells and the plates were
incubated at 37  C with 5% CO2 and 100% relative 2.5.2. In vivo PDT e±cacy
humidity for 4 h. After this time, the medium was When the tumor reached 5–7 mm in diameter, the
then discarded and 150
L DMSO was added to each mice were injected with TPMC at a dose of 8
M. At
well according to the method of Alley et al.35 The 24 h post-injection, the mice were restrained in plastic
MTT test was performed using an ELISA plate plexiglass holders without anesthesia and treated
reader at 570 nm (Bio-Rad, California, USA). with laser light (650 nm, 90 J/cm2, 50 mW/cm2). The
power was monitored during the entire treatment.
2.4.4. Phototoxicity Post-PDT, the mice were observed daily for tumor
regrowth or tumor cure. Visible tumors were
Cells were prepared as described above. Thereafter,
measured using two orthogonal measurements L and
the media containing TPMC were replaced with
W (perpendicular to L), and the volumes were cal-
drug-free medium containing 10% of serum and
culated using the Microsoft Excel formula V ¼
cells were irradiated at 650 nm at a °uence rate
LW 2/2 and recorded. Mice were considered as cured if
of 25 mW/cm2 and light doses ranging from 1.2 to
there was no palpable tumor by day 60–90. Once the
3.6 J/cm2. This wavelength was emitted by a Nd:
tumor reached 400 mm3, the mice were euthanized
YAG laser. Control experiments performed in the
due to tumor burden as per RPCI IACUC rules.
absence of any photosensitizer indicated that light
doses up to 3.6 J/cm2 caused no evident cell damage.
A plate similarly treated but not exposed to light was 3. Results
used as reference for the dark cytotoxicity in the
same experimental conditions. Experiments were 3.1. UV-Vis absorption spectrum
conducted in quadruplicate and repeated thrice. UV-Vis absorption spectrum was recorded on a stan-
Analysis of cell phototoxicity using the MTT assay as dard commercial spectrophotometer. The absorption

1540003-3
L.-J. Zhang et al.

TPMC
TPMC

Fluorescence (A.U.)
Absorption (A.U.)

550 600 650 700 750

300 400 500 600 700 800 wavelength (µm)
Wavelength (µm)
(a)
Fig. 2. UV-Vis absorption spectrum of TPMC in DMF. Its
maximum absorbance is at 420 nm, and at 527, 552, 600, 653 550
and 712 nm, also it has absorption. 3.260E+06

2.443E+06

Excitation wavelength (µm)

500
Table 1. Molar absorption coe±cients of TPMC.
1.625E+06

Molar absorption coe±cient 8.075E+05

Wavelength (nm) "ðM
1  cm
1 Þ 450

-10000

527 9091
552 7142 400
600 3703
653 16,667
712 2857 350

600 620 640 660 680 700 720 740 760 780
Emission wavelength (µm)
peak of TPMC in DMF is at 420 nm, and it also has
absorption at 527, 552, 600, 653 and 712 nm (see (b)
Fig. 2). Molar absorption coe±cients of TPMC are
Fig. 3. Emission spectrum and excitation and emission spec-
shown in Table 1. tra matrix of TPMC in DMF. (a) Emission spectrum of TPMC,
which was excited at 420 nm, and its peak was at 656 nm. (b)
The matrix of excitation and emission spectra (Ex: 300–550 nm,
3.2. Fluorescence spectrum Em: 600–780 nm).
Fluorescence spectra were measured using spectro-
°uorimeter described in Sec. 2. TPMC can be exci- The singlet oxygen quantum yield was measured
ted at 420 nm, and its emission was monitored at as follows: 2 mL DMF solution containing 20
M
wavelengths 656 nm (see Fig. 3). DPBF and 0.5
M TMC was placed in a sealed
quartz cuvette. The overall optical density of the
solution was kept below 1.5, to be able to rely on the
3.3. Singlet oxygen quantum yield Beer–Lambert law (linear proportionality between
Energy transfer between the triplet state of photo- absorbance and concentration). The laser power
sensitizers and ground state molecular oxygen leads was 5 mW to ensure about 5% DPBF decay per 10 s
to the production of singlet oxygen. There is an irradiation interval. The experiment was repeated
essentiality of high e±ciency of transfer of energy three times.
between excited triplet state of TPMC and ground

 ðSÞ ¼
 ðRÞk S IaT ðRÞ=k R IaT ðSÞ ð1Þ
state of oxygen to generate large amounts of singlet
oxygen, necessary for PDT. Ia ¼ I0 ð1
e
2:3AÞ ð2Þ

1540003-4
 Anti-tumor activities of a novel chlorin derivative for PDT

0.6

Methylene blue
TPMC

-ln[DPBF]/[DPBF]0
0.4
0s
10 s
Absorption (A.U.)

20 s
30 s
--- 0.2

0.0
350 400 450 0 10 20 30 40 50 60

wavelength (µm) Irradiation Time (s)

(a) (b)

Fig. 4. Photodecomposition of DPBF by 1O2 after irradiation of methylene blue, TPMC in DMF (monitoring the maximum
absorption of DPBF at 410 nm).

 was calculated on the basis of Eqs. (1) R > 0:99946), as shown in Fig. 4. The 1O2 quantum
and (2).21 Superscript S and R indicate the sample yield (
 Þ of TPMC in DMF was 48%.
and reference compound, respectively. Ia is de¯ned
as the total amount of light absorbed by the sensi-
3.4. Cellular uptake of TPMC in
tizers. A is the corresponding absorbance at ir-
radiation wavelength. After the data were plotted Eca-109 and SMMC-7721 cells
as
ln½DPBF=½DPBF0 versus irradiation time t, The uptake of a 5
M solution of TPMC was
straight lines were obtained for the sensitizers, and measured after incubating Eca-109 and SMMC-
the slope for each compound was obtained after 7721 cells for di®erent time periods in the dark. As
¯tting with a linear function (correlation coe±cient shown in Fig. 5(a), a rapid signi¯cant uptake of

SMMC-7721 Eca-109
- + - +

20

15
nM/cell

10

5
Eca-109
SMMC-7721

0
0 5 10 15 20 25 30
Time (h)

(a) (b)

Fig. 5. Time-dependent uptake of TPMC. (a) Eca-109 and SMMC-7721 cells were incubated for di®erent time periods in the dark
with a 5
M concentration of TPMC. (b) Intracellular location of TPMC was visualized by confocal microscopy after incubating Eca-
109 and SMMC-7721 cells for 24 h in the dark. The upper panel corresponds to phase contrast and lower panel to °uorescence images.

1540003-5
L.-J. Zhang et al.

TPMC was observed after 30 min exposure, reach- induced a signi¯cant amount of nonviable cells and,
ing a plateau after 4 h. A nucleus location of TPMC therefore, this concentration was not used for
with a typical red °uorescence emission was photo-treatments.
observed by confocal microscopy after 24 h incu- Figures 6(b) and 6(c) showed the changes in cell
bation by exciting at 420 nm and detecting the viability caused by TPMC at di®erent concen-
emission °uorescence at 656 nm [see Fig. 5(b)]. trations and light doses, as evaluated by MTT
assay. The surviving fraction of Eca-109 and
SMMC-7721 cells that were exposed to light with-
3.5. Cell dark- and photo-toxicity out TPMC pre-incubation was similar to that in
TPMC was tested at di®erent concentrations controls (data not shown). Low light doses (1.2 J/
(2 to 10
M) for 24 h without red light irradia- cm2) caused moderate damage even at the highest
tion [see Fig. 6(a)]. Survival of Eca-109 and SMMC- concentration tested. By raising the light dose to
7721 cells, as assessed by MTT assay, was greater 3.6 J/cm2, lethality over 50% was achieved but this
than 80% for concentrations of 6
M or less. treatment was still judged insu±cient. Cell viability
When concentration was 8
M, a slight increase in below 25% was observed when using TPMC at
cytotoxicity was detected. However, 10
M TPMC concentrations equal or less than 6
M and at

(a)

100 100
Eca-109 SMMC-7721
1.2 J
1.2 J
2.4 J
80 80 2.4 J
3.6 J
3.6 J
% cell viability

% cell viablity

60 60

40 40

20 20

0 0
0 2 4 6 8 0 2 4 6 8
concentration (µM) concentration (µM)

(b)

Fig. 6. (a) Dark toxicity of each concentration assayed. Data correspond to mean values  SD from at least three di®erent
experiments. B.C) Light dose-dependent e®ects on cell viability after incubation with TPMC (2 to 8
M) for 24 h, followed by
irradiation. Di®erent treatments produce signi¯cant e®ects on the survival of Eca-109 (b) and SMMC-7721 (c) cells. Surviving
fractions of cells correspond to mean  SD values from at least three di®erent experiments.

1540003-6
 Anti-tumor activities of a novel chlorin derivative for PDT

(c)

Fig. 6. (Continued )

maximum light dose (3.6 J/cm2), whereas treat- shown in Fig. 7, TPMC was mainly found in the
ments with 8
M plus 3.6 J/cm2 induced lethal nuclear membranes and nucleus co-localized with
e®ect. Balance between dark toxicity and photo- the blue °uorescence of the nucleus probe. Thus,
damage suggested that 6 or8
M TPMC and 3.6 J/ pink vesicles were visualized from the overlay of the
cm2 were suitable conditions for e®ective photo- red °uorescence from TPMC.
dynamic studies.
3.7. In vivo PDT e±cacy
3.6. Intracellular location of TPMC To determine the e®ects of TPMC on esophageal
The nucleus location of TPMC was evaluated by cancer in vivo, 1  10 6 Eca-109 cells were injected
confocal microscopy after incubating Eca-109 and into the axils of nude mice to form solid tumors
SMMC-7721 cells for 24 h and staining them with (100 mm3 in size). 200
L of 8
M TPMC was then
°uorescent dye (Hoechst 33342) for cell nucleus. As injected into the tail vein and 24 h later the tumor

(a) (b)

Fig. 7. Intracellular localization of TPMC and staining cell nucleus. SMMC-7721 (a) and Eca-109 (b) incubated with 5
M of
TPMC for 24 h were stained with Hoechst 33342 (0.5
g/mL, 30 min) and kept in the dark. Red °uorescence corresponds to TPMC
and blue °uorescence represents the signal for Hoechst 33342.

1540003-7
L.-J. Zhang et al.

was irradiated with 50 mW/cm2 laser light for 4. Discussion

10 min (90 J/cm2). PDT-treated tumors began to
An ideal photosensitizer should have absorption
shrink within ¯ve days and became dark, hardened
spectra at long wavelengths, which allows deeper
and dried over the course of the next 10 days and
tissue penetration and decreased nonspeci¯c
eventually formed a scab by 20 days after treat-
lesions.9 For instance, 630 nm light penetrates less
ment. The normal skin phototoxicity subsided
than 0.5 cm whereas 700 nm light reaches a depth of
within four days. In contrast, the tumors in control
near 0.8 cm.22 In the present research, we show how
group continued to grow and were signi¯cantly
photodynamic treatments a®ect cultured Eca-109
larger than in PDT-treated group after 10 days
and SMMC-7721 tumor cells with chlorin derivative
post-treatment [see Fig. 8(a)]. The animals were
TPMC (see Fig. 1). Both in vitro and in vivo date
monitored for 60 days for recurring tumor growth
supported that TPMC had the potential to be a
[see Fig. 8(b)]. TPMC-PDT gave more than 50%
photosensitizer for PDT.
tumor cure.
Di®erent wavelengths have di®erent tissue pen-
etrations.9,22 TPMC may be used for the treatment
3
of port wine stains (PWS), because of its absorption
at 527 and 552 nm (see Fig. 2). PWS appears in the
Control upper of dermis, requiring wavelengths from 488 nm
PDT
to 578.2 nm (tissue penetration: 0:2  0:4 cm) for
Turmor growth rate

2
treatment in PDT.23,24 The absorption at 653 and
712 nm are suitable for deeper tumors.
Besides many photosensitizers being bright
1
°uorophores, they also tend to emit light in the
near-infrared (NIR) portion of the spectra that is
useful for in vivo imaging.3,25,26 Excited by 420 nm
0
0 5 10 15 20 25 light, TPMC can emit red °uorescence at 656 nm
Days post-treatment (day) (see Fig. 3), which means it has the prospects to
(a) become a new reagent for targeting °uorescence
imaging in tumor diagnosis.27,28
Figure 5 demonstrated e±cient uptake of TPMC
Control
100
PDT
by Eca-109 and SMMC-7721 cells and its relation
% Mice with Tumor size<400 mm3

to incubation time. Fluorescence microscopy stud-

80
ies using the well-known nuclear °uorescent dye
% Tumor Cure

60
(Hoechst 33342) revealed that TPMC could ac-
cumulate in nuclear membranes and nucleus (see
40 Fig. 7). In this respect, it is already known that
subcellular localization of several porphycene de-
20
rivatives is heterogenous (lysosomes, mitochondria,
ER, Golgi apparatus, among others), as re°ected in
0
0 20 40 60 a recent review.29
Time (days)
There are numerous in vitro studies assessing the
(b) e®ectiveness of new PSs. However, use of many
Fig. 8. In vivo photosensitizing e±cacy of TPMC against
di®erent photodynamic protocols precludes com-
esophageal cancer in nude mice bearing Eca-109 tumors parison of the relative e±ciency of new photo-
(10 mice/group). The mice in PDT group were treated with activable drugs. In this regard, research carried out
laser light (650 nm, 90 J/cm2) at 24 h post-injection of the drug. by Berlanda et al.30 on A431 cell line with some of
The control mice were not subjected to any photosensitizer or the most used PSs (temopor¯n, hypericin, Photo-
light. (a) Tumor growth rate. The tumors in control group
frin, AlPcS4 and PPIX), provides a valuable refer-
continued to grow and were signi¯cantly larger than in PDT-
treated group after 10 days post-treatment. (b) Tumor cure ence for benchmarking purposes. Experimental
rate. At day 60, TPMC gave 60% tumor response (6/10 mice conditions of the present work are quite similar to
were tumor free). those of the photosensitizers mentioned above. This

1540003-8
 Anti-tumor activities of a novel chlorin derivative for PDT

includes incubation time with PS (18 versus 24 h), 81101298), Foundation of Shanghai government
light °uence (1.2–3.6 J/cm2) and time interval to (13431900700, 13430722300, 13ZR1441000,
perform MTT assay (24 h post-irradiation in both 13ZR1440900), Foundation of Donghua University
cases). (No. 11D10501; 12D10515) and Foundation of
TPMC induced no dark toxicity in the range of Yiwu Science and Technology Bureau (2011-G1-
concentrations used in the present photodynamic 15; 2012-G3-02).
studies. Cell survival after incubation with TPMC
and red-light irradiation is related to both the
TPMC concentration and light dose (see Fig. 6). It References
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