Mitochondria-targeting properties and photodynamic activities of porphyrin derivatives bearing cationic pendant

Article abstract of DOI:10.1016/j.jphotobiol.2009.12.003

Four meso-tetraphenylporphyrin derivatives bearing either triphenylphosphonium ion-(P1 and P2) or triethylammonium ion-(P3 and P4) terminated alkoxy group at either para-(P1 and P3) or meta-(P2 and P4) position of one meso-phenyl group were designed and s

Full text of DOI:10.1016/j.jphotobiol.2009.12.003

Journal of Photochemistry and Photobiology B: Biology 98 (2010) 167–171
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Mitochondria-targeting properties and photodynamic activities of porphyrin
derivatives bearing cationic pendant
*
Wanhua Lei, Jingfan Xie, Yuanjun Hou, Guoyu Jiang, Hongyan Zhang, Pengfei Wang, Xuesong Wang ,
Baowen Zhang
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2009
Received in revised form 8 December 2009
Accepted 14 December 2009
Available online 22 December 2009
Four meso-tetraphenylporphyrin derivatives bearing either triphenylphosphonium ion-(P1 and P2) or tri-
ethylammonium ion-(P3 and P4) terminated alkoxy group at either para-(P1 and P3) or meta-(P2 and P4)
position of one meso-phenyl group were designed and synthesized. P1–P4 show similar absorption and
fluorescence emission spectra and 1O2 quantum yields. The more lipophilic nature of triphenylphospho-
nium ion over triethylammonium ion renders P1 and P2 higher octanol/water partition coefficients than
P3 and P4. Confocal fluorescence microscopy proved that P1–P4 are all mitochondria-targeting. MTT
assay showed that P1–P4 presented significant phototoxicity at the concentrations that dark toxicity is
negligible towards human breast cancer cell line MCF-7 cells, displaying their application potential in
PDT.
Keywords:
Photodynamic therapy
Mitochondria-targeting
Lipophilic cation
Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction
localize to mitochondria are reported to be more efficient in killing
cells than those that localize at other cellular sites [14–16]. How-
Photodynamic therapy (PDT) is a novel treatment modality for
numerous cancers and certain noncancerous diseases [1]. It utilizes
the photoreactions mediated by photosensitizer, light, and oxygen
to generate reactive oxygen species (ROS), such as singlet oxygen
(1O2), superoxide anion radical (OÀ2 ), and hydroxyl radical (ÅOH),
and relies on the cytotoxic effects of ROS to lead to apoptosis or
necrosis of abnormal cells. The short lifetime and thus the very lim-
ited diffusion distance of ROS in biological systems, for example, the
ever, preparing mitochondria-targeting PDT photosensitizers by ra-
tional molecular design is still in its infancy [17–19]. Two strategies
are widely used to render biologically active molecules mitochon-
dria-targeting capability: (1) tethering a mitochondrial localization
peptide sequence, which utilizes protein import pathway; and (2)
attaching a lipophilic cation group, which utilizes the high mem-
brane potential (À180 mV) across the inner mitochondrial mem-
brane [20]. Recently, Asayama et al. [17] used a peptide modified
Mn-porphyrin to target mitochondria, Vicente and co-workers
[18] synthesized a series of amphiphilic cationic porphyrin deriva-
tives bearing either a group with positive charge (guanidinium or
biguanidinium) or a mitochondrial localization peptide, and Kope-
diffusion range of 1O2 is smaller than 0.1
lm in tissues, render PDT
‘‘in situ” character, i.e. the site of photodamage is also the site where
ROS is generated [2]. Consequently, the PDT efficacy is dependent
not only on the selective accumulation but also on the subcellular
localization of photosensitizers in abnormal cells. In this regard,
mitochondria-targeting is of importance for PDT due to its key role
in regulating apoptotic cell death [3]. So far two major apoptotic
pathways have been characterized: the death receptor-mediated,
or extrinsic pathway, and the mitochondria-mediated apoptosis,
or intrinsic pathway [4,5]. The central place of mitochondria as reg-
ulators of apoptotic cell death has stimulated great interest in
developing mitochondria-targeting chemotherapy [6–9]. In the
field of PDT, some photosensitizers, such as protoporphyrin IX
[10,11] and phthalocyanine Pc4 [12,13], have been known mito-
chondria localization for a long time, and photosensitizers that
ˇ
cek and co-workers [19] targeted a HPMA copolymer-bound Mce6
to mitochondria by grafting triphenylphosphonium ion. Though
there are a number of lipophilic cations can be chosen, triphenyl-
phosphonium ion is particularly unique for mitochondria targeting,
mainly due to its simplicity in structure and ease to be linked on the
target molecules. Thus, triphenylphosphonium ion-based com-
pound class includes the majority of the non-peptidic mitochon-
drial targeting agents synthesized to date [20]. So far, only one
example of triphenylphosphonium ion-modified PDT photosensi-
tizer was reported [19], in which the complicated synthesis hin-
dered in depth studies, such as the influence of linking modes
between porphyrin derivatives and triphenylphosphonium ion
and further structural optimization for PDT efficacy improvement.
In this work, four cation ion-modified meso-tetraphenylporphyrin
* Corresponding author. Tel.: +86 10 8254 3592; fax: +86 10 6255 4670.
E-mail addresses: g203@mail.ipc.ac.cn, xswang@mail.ipc.ac.cn (X. Wang).
1011-1344/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2009.12.003

168
W. Lei et al. / Journal of Photochemistry and Photobiology B: Biology 98 (2010) 167–171
derivatives (P1-P4) were synthesized through a facile way, two of
them (P1 and P2) are based on triphenylphosphonium ion and
the other two (P3 and P4) triethylammonium ion, and the cation
pendant is anchored on the phenyl group at either para- or meta-
position (Scheme 1). All the four porphyrins were found to specifi-
cally localize in mitochondria of MCF-7 cells and show good photo-
dynamic activities. In particular, P1 presents the highest cellular
uptake and the lowest dark toxicity among the four compounds,
implying the important role of modification mode. The results sug-
gest that PDT efficacy may be further improved via delicate design
of the linking mode of the lipophilic cation group to porphyrin-
based chromophores. Moreover, easy preparation method adopted
in our work make the structural optimization convenient.
8.20–8.22 (d, 6H, J = 6.4 Hz), 8.84 (bs, 8H). ESI-MS: m/z = 947.5
([M-Br]+).
5-(m-(4-triphenylphosphonium)-butoxyphenyl)-10,15,20-tri-
phenylporphyrin bromide (P2). 1H NMR (400 MHz, in CDCl3):
d = À2.79 (s, 2H), 1.93–1.95 (m, 2H), 2.30–2.33 (m, 2H), 4.00 (m,
2H), 4.28–4.31 (t, 2H, J = 5.5 Hz), 7.28–7.30 (d, 1H), 7.53–7.65 (m,
11H), 7.74–7.83 (m, 16H), 8.20–8.22 (d, 6H, J = 5.4 Hz), 8.82–8.85
(m, 8H). ESI-MS: m/z = 947.5 ([M-Br]+).
5-(p-(4-triethylammonium)-butoxyphenyl)-10,15,20-triphen-
ylporphyrin bromide (P3). 1H NMR (400 MHz, in CDCl3): d = À2.77
(s, 2H), 1.19–1.23 (t, 9H, J = 14.0 Hz), 2.00–2.20 (bs, 4H), 3.40–3.70
(bs, 8H), 4.33 (bs, 2H), 7.25–7,27 (d, 2H, mixed with CHCl3), 7.72–
7.78 (m, 9H), 8.11–8.13 (d, 2H, J = 6.7 Hz), 8.20–8.22 (d, 6H,
J = 6.6 Hz), 8.84–8.85 (bs, 8H). ESI-MS: m/z = 786.5 ([M-Br]+).
5-(m-(4-triethylammonium)-butoxyphenyl)-10,15,20-triphen-
ylporphyrin bromide (P4). 1H NMR (400 MHz, in CDCl3): d = À2.80
(s, 2H), 1.11–1.13 (t, 9H, J = 14.2 Hz), 1.86–1.89 (m, 4H), 3.05–3.08
(m, 6H), 3.14–3.20 (m, 2H), 4.16–4.18 (t, 2H, J = 5.4 Hz), 7.62 (t, 1H,
J = 8.0 Hz), 7.68 (bs, 1H), 7.83 (d, 1H, J = 8.0 Hz), 8.19–8.21 (d, 6H,
J = 5.8 Hz), 8.84–8.87 (m, 8H). ESI-MS: m/z = 786.5 ([M-Br]+).
2. Material and methods
2.1. Synthesis
The mixture of 0.2 g of 5-(4-hydroxy-phenyl)-10,15,20-triphen-
ylporphyrin, 1.5 g of anhydrous K2CO3, and 15 mL of 1,4-dibromo-
butane was heated at 150 °C for 2 h under N2 atmosphere. After
removal of 1,4-dibromobutane in vacuum, the residue was ex-
tracted with chloroform and the extract was subjected to column
chromatography on silica gel using chlorofo 5-(p-(4-bromo)-but-
oxyphenyl)-10,15,20-triphenylporphyrin in yield of 87%. The ob-
tained product was mixed with excess triphenylphosphine and
heated at 100 °C for 14 h under N2 atmosphere. After cooling to
room temperature, chloroform was added to dissolve the solid.
Column chromatography of the chloroform solution on silica gel
using chloroform/petroleum ether (50/50) as eluent removed tri-
phenylphosphine at first and then the use of chloroform/methanol
(93:7) as eluent gave the target photosensitizer P1 in yield of 74%.
Similar procedures were used to prepare P2–P4, all in similar good
yields.
2.2. Singlet oxygen quantum yields [21]
A series of 2 mL of air-saturated acetonitrile solutions contain-
ing 1,3-diphenylisobenzofuran (DPBF, 20 lM) and the examined
photosensitizer, of which the absorbance at 512 nm originating
from the absorption of the photosensitizer was adjusted to the
same (OD = 0.15), were separately charged into an opened 1 cm
path fluorescence cuvette and illuminated with light of 512 nm
(obtained from a Hitachi F-4500 Fluorescence Spectrophotometer).
The consumptions of DPBF were followed by monitoring its fluo-
rescence intensity decrease at the emission maximum
(kex = 445 nm) at different irradiation time. Meso-tetraphenylpor-
phyrin (TPP) was used as standard, whose 1O2 quantum yield
was determined to be 0.60 in air-saturated acetonitrile [22,23].
5-(p-(4-triphenylphosphonium)-butoxyphenyl)-10,15,20-tri-
phenylporphyrin bromide (P1). 1H NMR (400 MHz, in CDCl3):
d = À2.77 (s, 2H), 2.00–2.08 (bs, 2H), 2.40–2.60 (bs, 2H), 4.10–
4.30 (m, 2H), 4.39 (bs, 2H), 7.19–7.22 (d, 2H, J = 8.0 Hz), 7.74–
7.81(m, 18H), 7.93–7.98 (m, 6H), 8.08–8.10 (d, 2H, J = 8.0 Hz),
2.3. n-Octanol/water partition coefficients (log P)
Typically,
a
solution of photosensitizer (50
lM) in equal
volumes of 50
l
M PBS, pH 7.4 (1 mL) and 1-octanol (1 mL) was
sonicated for 30 min. After separation by centrifugation, 20 lL of
P+
P+
NH
N
N
O
NH
N
N
O
HN
HN
P
1
P
2
N+
N+
O
NH
N
NH
N
N
N
O
HN
HN
P
P
3
4
Scheme 1. Structures of the examined photosensitizers P1–P4 (all isolated as bromide salts).

W. Lei et al. / Journal of Photochemistry and Photobiology B: Biology 98 (2010) 167–171
169
1-octanol phase and 100
lL of water phase were taken and diluted
by DMSO to 2 mL and 1 mL, respectively. The amounts of the pho-
tosensitizer in each phase were determined by monitoring fluores-
cence intensity at 655 nm (418 nm excitation) on a Hitachi F-4500
Fluorescence Spectrophotometer. The results are the average of
three independent measurements.
2.4. Cell culture
All tissue culture media and reagents were obtained from Hy-
Clone, Thermo Scientific. MCF-7 cells were maintained in DMEM
containing 10% fetal bovine serum.
2.5. Cellular uptake
MCF-7 cells were plated at 2 Â 105 per well in a Nunc 48 well
plate and allowed to grow for 24 h. Photosensitizer stocks were pre-
pared in DMSO at the concentration of 1 mM and then diluted into
medium to the final working concentrations (the final concentra-
Fig. 1. Absorption (solid line) and fluorescence emission (dash line, kex = 420 nm)
spectra of P1 in DMSO.
tion of DMSO was less than 5%). The cells were exposed to 10 lM
of photosensitizer for 0, 1, 2, and 4 h, respectively. At the end of
3. Results and discussion
the incubation time, the loading medium was removed and the cells
3.1. Photophysical and photochemical properties
were washed with 500
lL of PBS three times. The cells were solubi-
lized upon addition of 500
l
L of 0.25% Triton X-100 in PBS. Fluores-
As expected, the four porphyrin derivatives exhibit nearly iden-
tical photophysical properties due to their high similarity in chem-
cence emission intensities at 655 nm (418 nm excitation) were
measured to determine the porphyrin concentration that taken
up, on a Hitachi F-4500 Fluorescence Spectrophotometer.
ical structures. Fig.
1 shows the absorption spectrum and
fluorescence emission spectrum of P1 in DMSO, in which one
strong Soret band, four less intense Q bands, as well as an emission
band with fine structures are observed, all typical for porphyrins
[24,25]. Similar absorption and fluorescence spectra were obtained
for P2–P3. The absorption maxima of the Soret bands and the
emission maxima of P1–P4 were collected in Table 1. The marginal
spectrum red shift (about 1 nm) for P1 versus P2 or for P3 versus
P4 originates from the substitution of alkoxy group at the different
positions (para- versus meta-) of the phenyl group.
Singlet oxygen quantum yields of P1–P4 in acetonitrile were
determined using 1,3-diphenylisobenzofuran (DPBF) as the chem-
ical trap of 1O2 [21], which does not fluoresce upon oxidation by
1O2, and meso-tetraphenylporphyrin (TPP) as reference [22,23],
whose 1O2 quantum yield in acetonitrile was measured to be 0.6.
Fig. 2 gives the DPBF fluorescence intensity as the function of irra-
diation time. The fluorescence quenching rates are proportional to
the 1O2 quantum yields of the photosensitizers. Thus, the 1O2
quantum yields of P1–P4 were measured to be 0.53, 0.50, 0.57,
and 0.58, respectively. Obviously, P1–P4 can generate 1O2 as effi-
ciently as their parent molecule TPP, and therefore are expected
to be effective PDT agents.
2.6. Subcellular localization
The MCF-7 cells were plated on Culture Dishes containing a
microscope slide for confocal laser scanning microscopy (CLSM)
observations. For the colocalization experiments the cells were
incubated concurrently with 10 lM of photosensitizer and 10 lM
of Rhodamine 123 for 4 h. Then the slides were washed three times
with 5 mM PBS pH 7.4. Fluorescent microscopy was performed
using a Nikon C1Si inverted fluorescent microscope and the magni-
fication employed was 10 Â 60.
2.7. Dark cytotoxicity
The MCF-7 cells were plated as described above and allowed
24 h to attach. The cells were exposed to increasing concentrations
of photosensitizer up to 50 lM and incubated for 24 h. The loading
medium was then removed and the cells were fed with medium
containing MTT from Sigma. Cell viability was then measured by
reading the absorbance at 570 nm using a Thermo MK3 Multiscan
microplate reader. The signal was normalized to 100% viable
(untreated) cells.
Though the porphyrins bearing either triphenylphosphonium
ion or triethylammonium ion exhibit similar absorption spectra
as well as 1O2 quantum yields, their amphiphilicity, a property also
Table 1
Absorption and emission maxima, 1O2 quantum yields, and n-octanol/water partition
coefficients (log P) for P1–P4.
2.8. Phototoxicity
Porphyrins
P1
P2
P3
P4
The MCF-7 cells were prepared as described above and treated
with photosensitizer at the concentrations of 0, 1, 2, 4, 6, 8, and
10 lM, respectively. The cells were then exposed to light
>550 nm (using an Oriel 91192 Solar simulator as light source
and a 550 nm cut-off filter to remove the short wavelength light)
for 30 min. The irradiation intensity was about 14 mW/cm2 and
the total light dose was approximately 25 J/cm2. The cells were
returned to the incubator overnight and assayed for viability as
described above.
(nm)a
(nm)b
ab
420
419
420
419
k
k
max
em
658
0.53
1.57
657
0.50
1.87
657
0.57
0.73
656
0.58
0.84
max
(/1O2)c
Log Pd
a
b
C
d
Absorption maximum in DMSO.
Fluorescence emission maximum in DMSO.
1O2 quantum yield in acetonitrile, using TPP as reference (0.60).
SD < 5%.

170
W. Lei et al. / Journal of Photochemistry and Photobiology B: Biology 98 (2010) 167–171
acter of P2 than P1 as well as P4 than P3 suggests that substitution
position of cationic pendant on the phenyl group of porphyrins also
play a delicate role in the amphiphilicity.
3.2. Cellular uptake
The time-dependent cellular uptake of P1–P4 was evaluated in
human breast cancer cell line MCF-7 at a concentration of 10 lM,
and the results are shown in Fig. 3. P3 and P4, bearing triethylam-
monium ion-based pendant, exhibit lower cell uptake than P1 after
4 h incubation. Because the process of partitioning has not reached
equilibrium during 4 h, the differences in cellular uptake are most
likely related to kinetics of binding rather than to the extent of
binding. The observed faster cellular uptake of P1 may presumably
attributed to its higher lipophilic character, which facilitates it to
penetrate into the bilayer membrane of cells [26,27].
Interestingly, P2, which is more lipophilic than P1, was taken up
just in the same amount as P3 and P4, nearly half of that of P1.
Fig. 2. DPBF consumption as a function of irradiation time in air-equilibrated
CH3CN solutions of TPP (Square), P1 (up triangle), P2 (down triangle), P3 (left
triangle), and P4 (right triangle). I0 and It are, respectively, the fluorescence
intensities before and after irradiation.
3.3. Subcellular localization
The four cationic porphyrins P1–P4 show similar fluorescence
patterns in MCF-7 cells when investigated by confocal fluorescence
microscopy. Fig. 4 shows the confocal micrographs of the double-
stained MCF-7 cells with P1 and a mitochondria specific fluorescent
probe Rhodamine 123 [20]. When excited at 405 nm, red fluores-
cence attributable to P1 emission was observed (left panel in
Fig. 4), while 488 nm of excitation gave rise to typical green fluores-
cence of Rhodamin 123 (middle panel). Their overlapping images
exhibited an orange color (right panel). A perfect superposition be-
tween the Rhodamine dye image and that of P1 is evident, proving
the subcellular localization of P1 in mitochondria. This was ex-
pected since both dyes are lipophilic cations, and thus have a pref-
erence for mitochondrial organelle, driven by the extra large
membrane potential across the inner mitochondrial membrane.
The same subcellular localization was also confirmed for P2–P4.
3.4. Cytotoxicity and phototoxicity
The dark cytotoxicity of the four porphyrins P1–P4 were evalu-
ated in MCF-7 cells by exposure to increasing amount of each por-
phyrin derivative for 24 h, and cell survival was determined by
MTT assay (Fig. 5). None of P1–P4 displayed significant dark cyto-
Fig. 3. Time-dependent uptake of P1 (up triangle), P2 (down triangle), P3 (left
triangle), and P4 (right triangle) at 10 lM by MCF-7 cells.
important for PDT [26,27], presents large differences. Table 1 shows
toxicity up to a concentration of 10
lM. The cytotoxicity emerged
the octanol/water partition coefficients of P1–P4 measured with pH
clearly at the concentrations higher than 10
l
M. P3 and P4 with
7.4 PBS (5 lM) as the aqueous phase. While Log P values of P1 and
triethylammonium ion pendant are significantly more cytotoxic
than P1 and P2 that bear triphenylphosphonium ion pendant. It
is worth to note that P1, which possesses the highest cell uptake
among the four porphyrins, show the least dark cytotoxicity. For
P2 are 1.57 and 1.87, the values for P3 and P4 are only 0.73 and
0.84, indicating somewhat stronger hydrophilic feature of P3 and
P4 than P1 and P2. The result demonstrates that triphenylphospho-
nium ion is a more lipophilic cation ion than its counterpart
triethylammonium ion. Moreover, a little bit higher lipophilic char-
example, at 25
lM, cell survival of P1 treated cells was 65%, in
sharp contrast to the case of P4, where the cell viability was only
Fig. 4. Confocal micrographs of the double-stained MCF-7 cells with P1 and Rhodamine 123 (each 10
lM incubated for 4 h). (a) P1 fluorescence image upon excitation at
405 nm, (b) Rhodamine 123 fluorescence image upon excitation at 488 nm, (c) overlay of the former two fluorescence images.

W. Lei et al. / Journal of Photochemistry and Photobiology B: Biology 98 (2010) 167–171
171
quantum yields, as well as mitochondria-targeting property. P1
exhibits the least dark cytotoxicity and P3 possesses the highest
phototoxicity among the four porphyrins, both toward MCF-7 cells.
Importantly, all of them present significant phototoxicity but neg-
ligible dark toxicity at the concentrations 6–10
their potential in PDT.
lM, displaying
Acknowledgement
This work was financially supported by NNSFC (20772133,
20873170) and CAS (KJCX2.YW.H08).
References
[1] M.R. Detty, S.L. Gibson, S.J. Wagner, Current clinical and preclinical
photosensitizers for use in photodynamic therapy, J. Med. Chem. 47 (2004)
3897–3915.
[2] M. Sibrian-Vazquez, T.J. Jensen, M.G.H. Vicente, PorphyrinÀretinamides:
synthesis and cellular studies, Bioconjugate Chem. 18 (2007) 1185–1193.
[3] N.L. Oleinick, R.L. Morris, I. Belichenko, The role of apoptosis in response to
photodynamic therapy: what, where, why, and how, Photochem. Photobiol.
Sci. 1 (2002) 1–21.
Fig. 5. Dark toxicity of P1 (up triangle), P2 (down triangle), P3 (left triangle), and P4
(right triangle) at varied concentrations toward MCF-7 cells using MTT assay.
[4] Y. Shi, A structural view of mitochondria-mediated apoptosis, Nat. Struct. Biol.
8 (2001) 394–401.
[5] R.D. Almeida, B.T. Manadas, A.P. Carvalho, C.B. Duarte, Intracellular signaling
mechanisms in photodynamic therapy, Biochim. Biophys. Acta 1704 (2004)
59–86.
[6] J.L. Hickey, R.A. Ruhayel, P.J. Barnard, M.V. Baker, S.J. Berners-Price, A.
Filipovska, Mitochondria-targeted chemotherapeutics: the rational design of
gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer
cells and target protein selenols in preference to thiols, J. Am. Chem. Soc. 130
(2008) 12570–12571.
[7] D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death,
Science 305 (2004) 626–629.
[8] L. Galluzzi, N. Larochette, N. Zamzami, G. Kroemer, Mitochondria as
therapeutic targets for cancer chemotherapy, Oncogene 25 (2006) 4812–4830.
[9] V.R. Fantin, P. Leder, Mitochondriotoxic compounds for cancer therapy,
Oncogene 25 (2006) 4787–4797.
[10] A. Verma, J. Nye, S.H. Snyder, Porphyrins are endogenous ligands for the
mitochondrial (peripheral-type) benzodiazepine receptor, Proc. Natl. Acad. Sci.
USA 84 (1987) 2256–2260.
[11] A. Verma, S.L. Facchina, D.J. Hirsch, S.Y. Song, L.F. Dillahey, J.R. Willams, S.H.
Snyder, Photodynamic tumor therapy: mitochondrial benzodiazepine
receptors as a therapeutic target, Mol. Med. 4 (1998) 40–45.
[12] J. Morgan, A.R. Oseroff, Mitochondria-based photodynamic anti-cancer
therapy, Adv. Drug Delivery Rev. 49 (2001) 71–86.
[13] N.S. Trivedi, H.W. Wang, A.L. Nieminen, N.L. Oleinick, J.A. Izatt, Quantitative
analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label
confocal fluorescence microscopy, Photochem. Photobiol. 71 (2000) 634–639.
[14] D. Kessel, Y. Luo, Mitochondrial photodamage and PDT-induced apoptosis, J.
Photochem. Photobiol. B: Biol. 42 (1998) 89–95.
[15] D. Kessel, Y. Luo, Y. Deng, C.K. Chang, The role of subcellular localization in
initiation of apoptosis by photodynamic therapy, Photochem. Photobiol. 65
(1997) 422–426.
[16] D. Kessel, Y. Luo, Photodynamic therapy: a mitochondrial inducer of apoptosis,
Cell Death Differ. 6 (1999) 28–35.
[17] S. Asayama, E. Kawamura, S. Nagaoka, H. Kawakami, Design of manganese
porphyrin modified with mitochondrial signal peptide for a new antioxidant,
Mol. Pharm. 3 (2006) 468–470.
Fig. 6. Phototoxicity of P1 (up triangle), P2 (down triangle), P3 (left triangle), and
P4 (right triangle) at varied concentrations toward MCF-7 cells using MTT assay.
25%. The dark cytotoxicity follows the order of P4 > P3 > P2 > P1 at
the examined concentrations.
For phototoxicity study, cultured cells were incubated for 4 h in
the dark with various concentrations of photosensitizer and then
irradiated with broadband light (>550 nm) for 30 min. After irradi-
ation, the cells were incubated for another 19.5 h and then sub-
jected to cell survival analysis. Photosensitizer-free controls (i.e.
similar procedures except that no photosensitizer was added) were
also carried out. The data presented in Fig. 6 demonstrate that P3
photodamage MCF-7 cells with somewhat higher efficiency among
[18] M. Sibrian-Vazquez, I.V. Nesterova, T.J. Jensen, M.G.H. Vicente, Mitochondria
targeting by guanidineÀ and biguanidineÀporphyrin photosensitizers,
Bioconjugate Chem. 19 (2008) 705–713.
ˇ
ˇ
[19] V. Cuchelkar, P. Kopecková, J. Kopecek, Novel HPMA copolymer-bound
constructs for combined tumor and mitochondrial targeting, Mol. Pharm. 5
(2008) 776–786.
the four porphyrins. Under the concentration of 2 lM, P3 reduced
cell viability by 80%, while the other three porphyrins gave a 30–
40% reduction in cell viability. At the concentrations equal to or lar-
[20] A.T. Hoye, J.E. Davoren, P. Wipf, M.P. Fink, V.E. Kagan, Targeting mitochondria,
Acc. Chem. Res. 41 (2008) 87.
[21] R.H. Young, K. Wehrly, R.L. Martin, Solvent effects in dye-sensitized
photooxidation reactions, J. Am. Chem. Soc. 93 (1971) 5774–5779.
[22] F. Wilkinson, Quantum yields for the photosensitized formation of the lowest
electronically excited singlet state of molecular oxygen in solution, J. Phys.
Chem. Ref. Data 22 (1993) 113–262.
[23] R.W. Redmond, J.N. Gamlin, A compilation of singlet oxygen yields from
biologically relevant molecules, Photochem. Photobiol. 70 (1999) 391–475.
[24] M. Gouterman, Spectra of porphyrins, J. Mol. Spectrosc. 6 (1961) 138–163.
[25] M. Gouterman, G.H. Wagni’ere, L.C. Snyder, Spectra of porphyrins part II. Four
orbital model, J. Mol. Spectrosc. 11 (1963) 108–127.
ger than 6 lM, the four porphyrins showed similarly effective
phototoxicity, indicating their potential in PDT.
4. Conclusions
In summary, four meso-tetraphenylporphyrin derivatives
bearing either triphenylphosphonium ion (P1 and P2) or triethyl-
ammonium ion (P3 and P4) terminated alkoxy group at either
para- (P1 and P3) or meta- (P2 and P4) position of one meso-phenyl
group were designed and synthesized. It was found that P1–P4
show similar absorption and fluorescence emission spectra, 1O2
[26] T.J. Dougherty, Photosensitizers: therapy and detection of malignant tumors,
Photochem. Photobiol. 45 (1987) 879–889.
[27] R.W. Boyle, D. Dolphin, Structure and biodistribution relationships of
photodynamic sensitizers, Photochem. Photobiol. 64 (1996) 469–485.