The synthesis and photodynamic properties of meso-substituted, cationic porphyrin derivatives in HeLa cells

Article abstract of DOI:10.1016/j.dyepig.2009.07.008

A series of asymmetric porphyrins with varying substituents, such as 4-hydrophenyl and N-methyl-4-pyridyl, were synthesized and characterized and their cell uptake, intracellular localization, cytotoxicities and phototoxicities were evaluated in vitro. The most phototoxic of the porphyrins synthesized, 5,10-di-(N-methyl-4-pyridyl)-15,20-(4-hydroxyphenyl)-21,23H-porphyrin, which was mainly localized in the mitochondria and displayed low levels of dark toxicity toward human cancer HeLa cells, offers potential application in photodynamic therapy.

Full text of DOI:10.1016/j.dyepig.2009.07.008

Dyes and Pigments 84 (2010) 140–147
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Dyes and Pigments
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The synthesis and photodynamic properties of meso-substituted, cationic
porphyrin derivatives in HeLa cells
,d,
Cheng-Liang Peng a, Ping-Shan Lai b, Cheng-Chung Chang b, Pei-Jen Lou c, Ming-Jium Shieh a
*
a Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan
b Department of Chemistry, National Chung Hsing University, No. 250, Kuo-Kuang Road, Taichung 402, Taiwan
c Department of Otolaryngology, National Taiwan University Hospital and College of Medicine, #7, Chung-Shan South Road, Taipei 100, Taiwan
d Department of Oncology, National Taiwan University Hospital and College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of asymmetric porphyrins with varying substituents, such as 4-hydrophenyl and N-methyl-4-
pyridyl, were synthesized and characterized and their cell uptake, intracellular localization, cytotoxicities
and phototoxicities were evaluated in vitro. The most phototoxic of the porphyrins synthesized, 5,10-
di-(N-methyl-4-pyridyl)-15,20-(4-hydroxyphenyl)-21,23H-porphyrin, which was mainly localized in the
mitochondria and displayed low levels of dark toxicity toward human cancer HeLa cells, offers potential
application in photodynamic therapy.
Received 22 December 2008
Received in revised form
9 July 2009
Accepted 14 July 2009
Available online 22 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Photodynamic therapy
Cancer
Synthesis
Asymmetric porphyrins
Mitochondria
Partition coefficients
1. Introduction
Three fundamental requirements for PDT are oxygen, light
source, and photosensitizer. Each factor is harmless by itself, but
Photodynamic therapy (PDT) is an effective single modality
treatment for several medical indications, including cancer, age-
related maculopathy, periodontitis, malignant, and pre-malignant
skin disorders [1–3]. PDT involves the uptake of the photosensitizing
compound (photosensitizer) by cancerous tissues or other sites of
therapeutic interest (e.g. neovascular regions), followed by selective
irradiation with visible or IR light of an appropriate wavelength that
is absorbed by the photosensitizer [4–6]. The advantages of PDT are
that it is a precisely targeted treatment that utilizes selective
illumination that can be repeated in the same site if necessary, and it
is less invasive than surgery. Despite significant advantages, bio-
distribution of photosensitizers is limited. In addition phototoxicity
to the skin, due mainly to the hydrophobicity and non-selectivity of
PSs, is another considerable limitation [7].
their combination can produce cytotoxic agents. In PDT, the
photosensitizer preferentially localizes in rapidly growing cells and
gets activated by the exposure of light in presence of oxygen to
generate reactive oxidative species such as singlet oxygen (O*2) as
well as other ROS to kill tumor cells [8]. Due to its short lifetime,
singlet oxygen intracellular diffusion distance is not more than
0.01–0.02 mm and therefore its direct action is limited to the
intracellular structure where the sensitizer localizes [9,10]. More-
over, it is now recognized that damage to certain subcellular
structures is correlated with a specific cell death mechanism.
An ideal photosensitizer should have selectivity to tumor cells
so as to provide a basis for selectively killing tumor cell. Mito-
chondrial localized photosensitizers are able to induce apoptosis
very rapidly [11] and cytochrome c release is one of the best-known
apoptotic events after photosensitization [12,13]. The mitochon-
drion, due to its pivotal role in arbitrating cell apoptosis [14–16],
has recently been considered as a novel pharmacological target for
various clinical applications including cancer therapy [17,18].
Mitochondrial dysfunction leads not only to the interruption of the
energy supply, but also to the activation of the mitochondria
mediated apoptotic pathway [19].
* Corresponding author at: Institute of Biomedical Engineering, College of
Medicine and College of Engineering, National Taiwan University, No. 1, Section 1,
Jen-Ai Road, Taipei 100, Taiwan. Tel.: þ886
2
23123456x81444; fax: þ886
2
23912749.
E-mail address: soloman@ntu.edu.tw (M.-J. Shieh).
0143-7208/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2009.07.008

C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
141
Since the first approval of hematoporphyrin derivative (HpD) as
2.2. Synthesis of meso-substituted Porphyrins 1–5
a photosensitizer for PDT clinical applications [20], a large number
of porphyrin derivatives have been synthesized and investigated
for application in PDT; these include meso-tetraarylporphyrins,
porphyrin dendrimers, core modified porphyrins, chlorins, bacter-
iochlorins, and phthalocyanines [21–23].
The synthetic method used in our study was the Adler-Longo
method [26]. Adler and Longo modified the Rothemund reaction by
allowing benzaldehyde and pyrrole to react for 50 min in refluxing
propionic acid (141 ꢀC) in the presence of air. These comparatively
mild reaction conditions allowed a wider selection of substituted
benzaldehydes to be converted to the corresponding porphyrins in
yields of up to about 5%. The reaction was also amenable to large-
scale syntheses and multigram quantities of many porphyrins have
been prepared.
At present there exists no clear correlation between the meso-
substituents of porphyrins and their selective accumulation and
phototoxicity in tumor cells. Nevertheless, subcellular localization
plays a major role in photodynamic efficacy. The cellular affinity
of a photosensitizer is governed by its amphiphilic character, which
is dependent on the regiochemical arrangement of hydrophobic
and hydrophilic meso-substituents in the structure. In this study,
we investigated how modifications in the meso-substitution
pattern would affect the properties of photosensitizers and tried to
develop a novel mitochondria-specific photosensitizer for selective
tumor cell killing. This work describes the synthesis of a series
of meso-substituted porphyrins (Fig. 1), 4-hydrophenyl, and N-
methyl-4-pyridyl 4-pyridyl, with varying degrees of hydrophobic/
hydrophilic substitutions as model compounds for localization
studies of photosensitizers and photodynamic activity in a human
carcinoma cell line. Although 5,10,15,20-Tetrakis-(N-methyl-4-
pyridyl)-21,23H-porphyrin (Porphyrin 1) and 5,10,15,20-Tetrakis-
(4-hydroxyphenyl)-21,23H-porphyrin (Porphyrin 5) are well known
compounds for photodynamic therapy [24,25], Porphyrins 2, 3, and
4 have not, hitherto, been shown to display selective accumulation
and phototoxicity in tumor cells.
General Procedure:
Freshly distilled pyrrole (5.6 mL, 0.08 mol), 4-hydroxy-
benzaldehyde (4.9 g, 0.04 mol), and pyridine-4-aldehyde (3.8 mL,
0.04 mol) were mixed and then refluxing reagent grade propionic
acid (200 ml) was added (Fig. 1). After refluxing for 50 min, the
solution was cooled to room temperature and filtered, and the filter
was washed thoroughly with water. After washing, the resulting
purple crystals were air dried, and finally dried in vacuo to remove
adsorbed acid.
The purification of the products was achieved by repeated
column chromatography on silica gel (0.04–0.063 mm, 230–400
mesh ASTM) using dichloromethane, n-hexane, and methanol
(20:10:1) as eluents. The order of porphyrins eluted down from the
column was according to the following sequence 1a, 2a, 3a, 3a-
isomer, 4a and then 5. The structural formulas of all compounds are
shown in Fig. 1. Methylation was then effected by mixing the
precursors with MeI in CHCl3 at room temperature and monitored
by TLC, which afforded the target compounds in quantitative yields
(>90%). The solvent was removed under reduced pressure and the
solid was obtained after washing with diethyl ether. All compounds
were characterized by UV, 1H NMR spectroscopy, and Mass
spectrometry.
2. Experimental
2.1. Materials and instruments
All reagent grade solvents, including methanol, ethanol,
n-hexane, dichloromethane (DCM), acetone, dimethyl sulfoxide
(DMSO), and tetrahydrofuran (THF) were obtained from Tedia Inc.
(Fairfield, OH, USA), and were dried before application. DCM and
THF were dried over CaH2 and distilled before use. 1,3-diphenyli-
sobenzofuran (DPBF), n-octanol, pyrrole, 4-hydroxybenzaldehyde,
pyridine-4-aldehyde (caution: irritant; forms explosive mixtures
with air at ambient temperatures), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT), propionic acid and other
chemicals were obtained from Aldrich (Milwaukee, WI, USA). 1H
2.2.1. 5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)-21,23H-
porphyrin (1)
1H NMR (300 MHz, ppm; dmso-d6): 9.45 (6H, d, o-py), 9.14 (8H,
s,
b
-pyrrole), 8.95 (8H, d, m-py), 4.70 (12H, s, CH3), ꢁ3.10 (2H, s,
R-pyrrole NH). UV/VIS lmax (H2O) [nm] (log 3): 419 (5.19), 517 (4.10),
552 (3.83), 585 (3.83), 646 (3.49). MS m/z 1203.6 (Mþ–4I) (1202.49
calculated for C44H42I4N8); found C 44.95, H 3.52, I 42.21, N 9.32.
2.2.2. 5-(4-Hydroxyphenyl)-10,15,20-tri-(N-methyl-4-pyridyl)-
21,23H-porphyrin (2)
NMR spectra were recorded on
a Brucker Avance-500 MHz
FT-NMR. HRMS were recorded on a Finnigan/Thermo Quest MAT
95XL mass spectrometers. UV/Vis and fluorescence spectra were
recorded on a Varian Cary 50 UV–vis Spectrophotometer and
Varian Eclipse spectrophotometer respectively.
1H NMR (300 MHz, ppm; dmso-d6): 9.44 (6H, d, o-py), 9.14 (8H,
s,
b-pyrrole), 8.93 (6H, d, m-py), 8.08 (2H, d, OH-phenyl-Ho), 7.21 (2
H, d, OH-phenyl-Hm), 4.70 (9H, s, CH3), ꢁ3.11 (2H, s, R-pyrrole NH).
UV/VIS lmax (H2O) [nm] (log 3): 424 (5.17), 521 (3.79), 561 (3.31),
586 (3.30), 646 (2.97). MS m/z 1076.86 (Mþ–3I) (1075.56 calculated
for C45H40I3N7O); found C 50.25, H 3.75, I 35.40, N 9.12, O 1.49.
4
N
R1
H
+
1.propionic acid
141¢CX, 50min
(Z)
2. MeI, CHCl3
r.t., 6 h
2.2.3. 5,10-Di-(N-methyl-4-pyridyl)-15,20-(4-hydroxyphenyl)-
21,23H-porphyrin (3)
O
NH N
2
R2
1-5
1H NMR (300 MHz, ppm; dmso-d6): 9.45 (4H, d, o-py), 9.15 (8H,
R4
N
HN
+
s,
b-pyrrole), 8.95 (4H, d, m-py), 8.08 (4H, d, OH-phenyl-Ho), 7.21 (4
(Z)
H, d, OH-phenyl-Hm), 4.70 (6H, s, CH3), ꢁ3.10 (2H, s, R-pyrrole NH).
O
2
N
R3
UV/VIS lmax (H2O) [nm] (log 3): 426 (5.29), 523 (3.94), 566 (3.63),
593 (3.51), 655 (3.33). MS m/z 935.23 (Mþ–2I) (932.59 calculated
for C44H34I2N6O2); found C 56.67, H 3.67, I 27.22, N 9.01, O 3.43.
1a - 4a, 5
No.
1
R1=R2=R3=R4 : P
R=
P=
2
R1=R2=R3: P
R1=R2 : P
R4 : R
2.2.4. 5-(N-Methyl-4-pyridyl)-10,15,20-tri-(4-hydroxyphenyl)-
21,23H-porphyrin (4)
3
R3=R4 : R
N+CH3I-
4
R1 : P
R2=R3=R4: R
1H NMR (300 MHz, ppm; dmso-d6): 9.45 (2H, d, o-py), 9.15 (8H,
5
R1=R2=R3=R4: R
s,
b-pyrrole), 8.95 (2H, d, m-py), 8.09 (6H, d, OH-phenyl-Ho), 7.23 (6
Fig. 1. Synthesis of meso-substituted porphyrin analogues 1–5.
H, d, OH-phenyl-Hm), 4.70 (3H, s, CH3), ꢁ3.09 (2H, s, R-pyrrole NH).

142
C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
UV/VIS lmax (H2O) [nm] (log
3
): 423 (4.89), 518 (4.63), 553 (3.32),
porphyrins in medium to probe dose-dependent drug accumula-
tion. After incubation for 24 h, the cells were washed with PBS and
solubilized in 100 mL of 0.25% Triton X-100 in PBS. The cellular
595 (2.45), 648 (2.86). MS m/z 803.5 (Mþ–I) (805.66 calculated for
C44H32IN5O3); found C 65.59, H 4.00, I 15.75, N 8.69, O 5.96.
uptake of porphyrins was measured by determining the fluores-
cence emission of the accumulated porphyrins with Multiwell
Fluorscence Plate Reader (Millipore CytoFluor 2300) using excita-
tion/emission wavelengths of Soret band and 670 nm, respectively.
2.2.5. 5,10,15,20-Tetrakis-(4-hydroxyphenyl)-21,23H-porphyrin (5)
1H NMR (300 MHz, ppm; dmso-d6): 9.15 (8H, s,
b
-pyrrole), 8.09
(8H, d, OH-phenyl-Ho), 7.23 (8 H, d, OH-phenyl-Hm), ꢁ3.10 (2H, s,
R-pyrrole NH). UV/VIS lmax (THF) [nm] (log 3): 417 (5.55), 512 (4.24),
551 (4.04) 590 (3.88) 643 (3.53). MS m/z 668.5 (Mþ-I) (678.73
2.7. Cytotoxicity and phototoxicity of porphyrins
calculated for C44H30N4O4); found C 77.86, H 4.46, N 8.25, O 9.43.
To assess the cytotoxicity of the porphyrins, HeLa cells were
seeded on a 96-well culture plate at 3000 cells per well in a 100 mL
2.3. Partition coefficients
culture medium. The cells were allowed to settle and attached for
24 h. For dark cytotoxicity experiments, porphyrins were added to
Lipophilic and hydrophilic properties were characterized by the
partition coefficient Po/w ¼ Co/Cw of each compound between two
immiscible solvents n-octanol (o) and water (w) [27]. Partition
triplicate wells in 5-fold serial dilutions ranging from 100
0.8 M and incubated for 24 h. Phototoxicity experiments were
performed with HeLa cells in 96-well plates as described above,
with porphyrin concentrations of 100, 50, 20, 4, 0.8, and 0
mM to
m
coefficients were determined by adding 20 mM of sensitizer to 5 ml
octanol and then adding an equal volume of water. After each phase
was presaturated with the other, the tubes were vortexed for 1 min
at high speed, placed in a shaker for 4 h at room temperature, and
then centrifuged for 10 min at approximately 500 g to separate the
octanol and water phases. The phase containing the larger quantity
of partitioned compound (octanol) was determined and the parti-
tion coefficient P was characterized.
m
M
respectively. After 24 h incubation, the compounds were removed,
the cells washed with PBS and then exposed to light from
LumiSourceÒ (peak wavelength 435 nm, 13.5 mW/cm2) for 200 s.
The total light dose was 1.4 J/cm2. Cell survival was measured by
MTT assay. At 24 h post irradiation, 100 mL of MTT (final concen-
tration 0.5 mg/ml) was added to each well and incubated for 4 h at
37 ꢀC. Then the culture medium was replaced with 100 ul/well
DMSO to dissolve the blue formazan crystals produced by mito-
chondrial hydrogenases in the living cells. The 96-well culture plate
was shaken for 15 min at room temperature and read for optical
density (OD) immediately at 570 nm on a 318MC ELISA reader
(Sanke Instrument Co). The cell survival was expressed by the
absorbance changes, and the survival rate was given as the percent
ratio of absorbance of treated cells vs. control cells.
2.4. Singlet oxygen quantum yields
To evaluate the photosensitizing efficacy of the porphyrins, the
singlet oxygen quantum yields (F6) were measured by a steady-
state method using 1,3-diphenylisobenzofuran as the scavenger
[28]. In this procedure a solution of sensitizer and the 1,3-diphe-
nylisobenzofuran singlet oxygen acceptor was illuminated by
a light waveband centered at 652 nm which was absorbed only by
the sensitizer. The rate of consumption of 1,3-diphenylisobenzo-
furan was followed spectrophotometrically (Varian Cary 50 UV–Vis
spectrophotometer) by observing the decrease of an absorption
band at 410 nm as a function of irradiation time. Tetra (4-hydrox-
yphenyl) porphyrin (p-THPP, F6 ¼ 0.56 in methanol) was used as
the standard for the determination of absolute quantum yields [29].
In order to improve the accuracy in comparing the quantum effi-
ciencies of the sensitizers, each sensitizer was evaluated under
similar experimental conditions. In general, 2.5 mL of methanol
2.8. Intracellular localization of porphyrins
Cells were grown on glass slides in 35 mm petri dishes and
incubated for 24 h with porphyrins. Cells were washed with PBS
and examined directly or after co staining with 100 nM MitoTracker
Green FM (M-7514, Molecular Probes Inc, Eugene, OR) for 45 min at
37 ꢀC. Cells were examined with a Leica-SP2 laser scanning confocal
microscope (LSCM) (Leica Microsystems Heidelberg GmbH,
Heidelberg, Germany). Excitation light was 488 nm for all probes.
Fluorescence of the organelle probes was detected with an
emission filter of 510–550 nm and porphyrins fluorescence was
detected with an emission filter of 640–660 nm.
containing 1.0 mL of acetic acid was added to adequate amount of
sensitizer to give an optical density of 1.00 ꢂ 0.02 at its Soret
band. An aliquot of freshly prepared 1,3-diphenylisobenzofuran
in acetonitrile (w25 mL) was added to give an optical
density ¼ 1.00 ꢂ 0.02 at 410 nm. This solution was illuminated by
a 652 nm biolitec PDT laser (lmax ¼ 652 nm).
2.9. Statistical analysis
2.5. Cell culture and incubation conditions
Mean standard deviation and graphs were used to describe the
data. All p values were two-sided and their significance level was
0.05. SPSS 11.0 was used to conduct all statistical analysis.
Human cervical epithelioid carcinoma cells (HeLa) were main-
tained in a humidified 5% CO2 incubator at 37 ꢀC in MEM (Gibco
BRL, USA) supplemented with 10% heat-activated fetal bovine
serum (FBS) and 1% antibiotics (Antibiotic–Antimycotic, Gibco BRL,
USA). When cells were in the exponential growth phase, the
medium was removed and washed twice with PBS, and then cells
were replaced with fresh medium. Procedure to subculture HeLa
cells 2–3 times every week.
3. Results
3.1. Chemical characterization of several asymmetric porphyrins
3.1.1. Singlet oxygen quantum yields (F6
)
2.6. Cellular uptake
It was found that all the compounds were excellent singlet
oxygen generators with quantum yield (F6) values ranging from
0.25 to 0.64 in methanol (Table 1). Porphyrin 2 displayed the
highest quantum yield for singlet oxygen generation (0.64),
compared to the other porphyrin derivatives.
HeLa cells were seeded on 96-well culture plates at 3000 cells
per wells in a 100
mL culture medium. After growing overnight, cells
were exposed to various concentrations (0.8–100
mM) of different

C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
143
Table 1
Photophysical properties of meso-substituted porphyrins.
No.
Compound R1wR4
Absorption lmax in nm (log
3
)
Fluorescence lmax
Singlet oxygen
Partition
coefficients
(log P)
(nm)a
quantum yields
b
(F6
)
B (0,0)
Qy (1,0)
Qy (0,0)
Qx (0,1)
Qx (0,0)
Q (0,0)
Q (0,1)
In MeOH PyM:PhOHc
1
2
3
4
5
4:0
3:1
2:2
1:3
0:4
418 (4.39)
426 (4.87)
423 (5.15)
418 (54.8)
417 (5.55)
527 (3.45)
522 (3.90)
519 (4.05)
516 (3.69)
512 (4.24)
558 (3.18)
556 (3.75)
558 (3.85)
550 (3.28)
551 (4.04)
629 (3.28)
592 (3.66)
592 (3.71)
591 (2.55)
590 (3.88)
690 (3.36)
652 (3.44)
651 (4.51)
650 (2.76)
643 (3.53)
654
652
656
656
606
718
718
720
718
658
0.25
0.64
0.44
0.45
0.56
- 1.99
- 0.61
0.53
0.87
1.29
In H2O PyM:PhOHc
1
2
3
4
5
4:0
3:1
2:2
1:3
0:4
419 (5.19)
424 (5.17)
426 (5.29)
423 (4.89)
434 (4.91)
517 (4.10)
521 (3.79)
523 (3.94)
518 (4.63)
526 (2.99)
552 (3.83)
561 (3.31)
566 (3.63)
553 (3.32)
564 (3.88)
585 (3.83)
586 (3.30)
593 (3.51)
595 (2.45)
607 (3.26)
646 (3.49)
646 (2.97)
655 (3.33)
648 (2.86)
656
664
662
654
654
706
710
708
716
718
0.25
0.64
0.44
0.45
0.56
- 1.99
- 0.61
0.53
0.87
1.29
a
b
c
Excited at soret band.
Using Tetra (4-hydroxyphenyl) porphyrins (p-THPP, F6¼0.56 in methanol) as the reference.
PyM and PhOH are presented as N-methyl-4-pyridyl and hydroxyphenyl, respectively.
3.1.2. Partition coefficient
3.1.3. Absorbance and emission spectra
The degrees of lipophilicities of meso-substituted porphyrins
were determined by the partition coefficient values between
octanol and aqueous phase (Table 1). The porphyrins with greater
number of N-methyl-4-pyridyl substituents exhibited higher
hydrophilicity. However, the degrees of lipophilicities of meso-
substituted porphyrins increased as the number of 4-hydrophenyl-
substitutents increased.
The fluorescence emission and UV–visible spectral data of these
porphyrins in THF and H2O are shown in Fig. 2. The fluorescence
maxima show two bands (654–720 nm) upon the excitation by
Soret bands. Their UV–visible spectra consist of the intense Soret
band around 418–434 nm and weaker Q bands in the 510–660 nm
intervals with difference extinction coefficients in different solvent
(Table 1).
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
a
in H2O
0.40
b
in THF
1
2
1
2
3
4
5
0.35
0.30
3
4
5
0.25
0.20
0.15
0.10
0.05
0.00
400
500
600
700
400
500
600
700
nm
nm
1
2
3
4
5
in H2O
1
2
3
4
5
200
150
100
50
in THF
c
d
35
30
25
20
15
10
5
0
0
600
600
650
700
nm
750
800
650
700
nm
750
800
Fig. 2. UV–vis absorbance spectra of porphyrin derivatives 1–5 (1
(d). (lex ¼ soret band).
mM) in THF (a) and in H2O (b) Emission spectra of cationic porphyrin derivatives 1–5 (1 mM) in THF (c) and in H2O

144
C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
3.2. In vitro studies
4 showed a higher dark cytotoxicity probably as a result of the
higher cell uptake. The cytotoxicity of porphyrin derivatives
seemed to follow the partition coefficient values in the order of
5 > 4 > 2 > 3 >1 (Fig. 3 vs. Fig. 4).
However, those porphyrins were found to be phototoxic toward
human HeLa cells, upon exposure to low light dose (1.4 J/cm2), as
shown in Fig. 4(b). Lower concentrations of porphyrin derivatives
were also found to have a pronounced effect on cell viability.
Porphyrin 3 was the most phototoxic and the LD90 (light treatment
3.2.1. Cellular uptake of porphyrins
The cellular uptake was investigated in HeLa cells incubated in
the medium with the different porphyrin derivatives 1–5 respec-
tively for 24 h. Under similar conditions, the Porphyrins 2, 4, and 5
exhibited the highest uptake, and Porphyrin 1 had the lowest
uptake at either high or low concentration (Fig. 3). The uptake of
porphyrin derivatives were in the order of 5 ¼ 4 ¼ 2 > 3 > 1 at high
concentration (20
mM). Compared to the partition coefficients of
that results in a 90% cell kill) was about 3.1
Porphyrin (LD90 w 4.9 M) Porphyrin
Porphyrin 2 (LD90 w 20 M), and Porphyrin 1 (LD90 > 50
Although Porphyrin
mM, followed by
porphyrin derivatives (Fig. 3), their cellular uptake increased
proportionately with their corresponding partition coefficients
(log p). The porphyrins with lower partition coefficient had a lower
cellular uptake by the HeLa cells.
5
m
4
(LD90 w 6.9
m
m
M),
M).
m
2
exhibited the highest singlet oxygen
quantum yield of this series of porphyrins in MeOH, its phototox-
icity was lower than that of the other porphyrins. Porphyrins with
high hydrophilicity cannot easily diffuse across the cell membrane
and consequently have a lower cellular uptake. It was demon-
strated that Porphyrin 1 showed low levels of cell uptake resulting
in a lower cytotoxicity value and photodynamic efficiency.
3.2.2. Cytotoxicity and phototoxicity of porphyrins
The concentration-dependent dark cytotoxicity was investi-
gated in HeLa cells exposed to each porphyrin with different
concentrations (up to 100 mM) for 24 h in the dark. The dark
cytotoxicity of porphyrin derivatives increased proportionately
with the increase in porphyrin concentration. Porphyrins 5 and 4
showed higher dark cytotoxicity levels, whereas Porphyrin 1
exhibited a lower dark cytotoxicity value (Fig. 4). Porphyrins 5 and
The high phototoxicity of Porphyrin 3 despite its low cellular
uptake and relatively low singlet oxygen generating efficiency
Cytotoxicity
a
4µM
20µM
1
100
2
a
3
5
4
3
2
1
0
4
80
5
60
40
20
0
0
10
20
30
40
50
µ M
1
2
3
4
5
derivatives
Phototoxicity
b
1
2
3
4
5
2.0
1.5
b
100
80
60
40
20
0
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
0
10
30
40
50
20
µ M
1
2
3
4
5
derivatives
Fig. 4. (a) Drug dose-response curves obtained in HeLa cells following exposure to
different porphyrin derivatives for 24 h; and (b) irradiation with blue light (Lumi-
SourceÒ, 7 mW/cm2) for 200 s (1.4 J/cm2). The colorimetric MTT test was used for cell
death estimation.
Fig. 3. Comparison of cellular uptake (a) and partition coefficient (b) of a series of
porphyrin derivatives 1–5 at 4 and 20 M for 24 h by HeLa cell.
m

C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
145
could be explained by its specificity for mitochondrial localization
(Fig. 5). These results show that the degree of phototoxicity of
porphyrin derivatives correlates not only with their the ability for
generating singlet oxygen or the extent of their uptake into cells but
also on their specificity of subcellular localization.
3.2.3. Intracellular localization of porphyrins
In a comparative study with MitoTracker Green, the different
porphyrins showed distinct localizations in mitochondria. A repre-
sentative localization pattern of the most effective Porphyrins (3–5)
and the most ineffective Porphyrins (1 and 2) are shown in Fig. 5.
Fig. 5. Subcellular localization of porphyrin derivatives 1–5 in HeLa cells at 10 mM for 24 h. (a) MitoTracker Green fluorescence (b) porphyrin fluorescence (c) overlays of Mito-
Trackers with porphyrin fluorescence.

146
C.-L. Peng et al. / Dyes and Pigments 84 (2010) 140–147
Porphyrins 3–5 were found to localize in mitochondria, whereas
Porphyrin 2 had less colocalization with MitoTracker Green.
Porphyrin 2 (LD90 w 20 mM) and was less effective in vitro PDT, even
though its cellular uptake and ability for generating singlet oxygen
were high. As the result of confocal imaging, Porphyrin 2 exhibited
more aggregation in the cytosol, and Porphyrin 1 showed a low
fluorescence intensity due to its higher hydrophilicity and conse-
quently for its lower extent of cell uptake.
role in their cellular uptake and photocytotoxicity. In our study,
5,10-di-(N-methyl-4-pyridyl)-15,20-(4-hydroxyphenyl)-21,23H-
porphyrin (Porphyrin 3) showed a higher phototoxicity probably
due to its more specific mitochondrial localization, even though its
cellular uptake and singlet oxygen generating efficiency were not
the best in the series of porphyrin derivatives synthesized. Based on
the results of our study, this organelle-selective porphyrin can be
used as a basis for the synthesis of more compounds with improved
specificity and potency for potential anti-tumor therapy in the
future.
4. Discussion
An ideal photosensitizer should act on novel cellular targets that
are specific for cancer cells and sufficiently different from normal
cells so as to provide a basis for selective tumor cell killing. The
cellular affinity of a photosensitizer is governed by its amphiphilic
character, which is dependent on the regiochemical arrangement of
meso-substituents in the structure. In this study, we investigated
how the modification in the meso-substitution pattern would affect
the properties of porphyrin photosensitizers with the goal of
Acknowledgements
This research was supported by grants from National Science
Council of the Republic of China (NSC 97-2120-M-002-017).
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