A platinum functional porphyrin conjugate: An excellent cancer killer for photodynamic therapy

Article abstract of DOI:10.1246/bcsj.20180382

A novel tetracationic porphyrin-platinum(II) conjugate was synthesized and characterized. This complex, 4Pt(dach)ClTPyP, showed reasonable water solubility, lack of aggregation, and high singlet oxygen quantum yield. It also exhibited low dark cytotoxicity and excellent photocytotoxicity (Colon26: 0.17 μM; Sarcoma180: 0.25 M). The mechanisms of cell death have been investigated and are attributed to high singlet oxygen generation, internalization into nucleus, and a caspase-3 induced apoptosis pathway. In the in vivo photodynamic therapy (PDT) assay, 4Pt(dach)ClTPyP completely killed tumor tissue, not simply displaying inhibition of tumor growth, and no recurrence was seen 18 days later after a single administration. All these findings of 4Pt(dach)ClTPyP shed light on a potential clinical use for cancer PDT in the future.

Full text of DOI:10.1246/bcsj.20180382

Advance Publication Cover Page
A Platinum Functional Porphyrin Conjugate: An Excellent Cancer Killer for
Photodynamic Therapy
Xiaojun Hu,* Kazuma Ogawa, Siqiaozhi Li, Tatsuto Kiwada, and Akira Odani*
Advance Publication on the web January 24, 2019
doi:10.1246/bcsj.20180382
© 2019 The Chemical Society of Japan
Advance Publication is a service for online publication of manuscripts prior to releasing fully edited, printed versions. Entire
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Publication manuscripts.

A Platinum Functional Porphyrin Conjugate: An Excellent Cancer Killer for
Photodynamic Therapy
Xiaojun Hu,*1,2 Kazuma Ogawa,3 Siqiaozhi Li,2 Tatsuto Kiwada,4 and Akira Odani*4
1 Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China.
2 Division of Pharmaceutical Sciences, Graduate School of Medical Science, Pharmaceutical and Health Sciences, Kanazawa University,
Kanazawa 920-1192
3 Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192
4 College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192
E-mail: odani@p.kanazawa-u.ac.jp; xjhu11@shu.edu.cn
Akira Odani
Akira Odani received his Doctor's degree in pharmaceutical sciences in 1981 from Osaka University. He joined Professor
Yamauchi's group at Kanazawa University as Assistant Professor in 1981. He moved to the Department of Chemistry,
Nagoya University in 1987 and was promoted to Associate Professor in 1995, Associate Professor of Research Center for
Materials Science in 1999, Associate Professor of Graduate School of Sciences in 2006. In 2008, he moved to Kanazawa
University as Professor in Faculty of Pharmaceutical Sciences. His research interests include noncovalent interaction in
metal complex, solution chemistry of albumin-drug interaction, and platinum chemistry in medicine.
Xiaojun Hu
Xiaojun Hu received her Ph.D Degree in Pharmacy from Kanazawa University in 2017. After graduation, she became a
lecturer of the School of Life Science, Shanghai University. Her research work is mainly focused on platinum (II)-based
porphyrins for photodynamic therapy of cancer and surface plasmon resonance (SPR) and electrochemical biosensors for
disease diagnosis.
Abstract
A novel tetracationic porphyrin-platinum(II) conjugate
candidates to achieve a complete tumor removal by
platinum-based drugs coupled with photosensitizers.
was synthesized and characterized. This complex,
4Pt(dach)ClTPyP, showed reasonable water solubility, lack of
aggregation, and high singlet oxygen quantum yield. It also
exhibited low dark cytotoxicity and excellent photocytotoxicity
(Colon26: 0.17 μM; Sarcoma180: 0.25 μM). The mechanisms
of cell death have been investigated and are attributed to high
singlet oxygen generation, internalization into nucleus, and a
caspase-3 induced apoptosis pathway. In the in vivo
photodynamic therapy (PDT) assay, 4Pt(dach)ClTPyP
completely killed tumor tissue, not simply displaying inhibition
of tumor growth, and no recurrence was seen 18 days later after
a single administration. All these findings of 4Pt(dach)ClTPyP
shed light on a potential clinical use for cancer PDT in the
future.
Photodynamic therapy (PDT) is of particular significance
for noninvasiveness and high regional specificity. It combines
a photosensitizer, light source, and tissue oxygen that can lead
to the chemical destruction of some superficial and localized
cancers, as well as wet age-related macular degeneration.10-12
Under illumination, photosensitizers are activated and induce
energy transfer to produce highly toxic reactive oxygen species,
mainly singlet oxygen (1O2), which can kill cells by apoptosis
or necrosis.13 Hence, anticancer activity of photosensitizers
could be substantially improved by photoactivation. Porphyrins
and phthalocyanines are the representatives of photosensitizers
for PDT. They are thermodynamically and kinetically stable,
and show great retention or accumulation in tumors by
preferentially binding to low density lipoproteins.14,15 The
molecular structure of porphyrins is susceptible to be
opportunely functionalized by peripheral substituents, like
coordinating peripheral pyridylgroups on transition metal
complexes. The presence of metal complexes allows to modify
the local environment, thereby getting highly reactive species,
improving the solubility, or introducing specific functionalities.
But for the use of phthalocyanines, one of the main problems is
the self-aggregation in aqueous media, which may affect the
photosensitive activity. Therefore, porphyrins are a good
platform for PDT of tumor, as well as the introduction of
platinum complexes by conjugation reaction.
Keywords: Photodynamic therapy, Anticancer agent,
Porphyrin
1. Introduction
Platinum complexes, such as cisplatin, carboplatin, and
oxaliplatin [Pt(dach)(C2O4)], have been used as a first-line
therapy to treat colorectal cancer.1,2 Oxaliplatin proves to be
relatively easy to handle and well tolerated,3 though it is still
constrained by dose-limiting side-effects and acquired
resistance.4 As one of the most effective chemotherapeutic
agents in clinical use, the mechanism of tumor inhibition is
related to its high binding affinity of platinum(II) to nuclear
DNA.5 Recently, some researchers reported that the
combination of platinum complexes with photosensitizers can
provide enhanced tumor inhibition effects.6-9 Along these lines,
there is a growing need for developing new and excellent
There are a number of porphyrins in cancer PDT reported
to date. Most of them showed remarkable inhibition of tumor
growth in vitro and in vivo, but cannot thoroughly kill tumor
cells.6,7,16 On the other hand, the notorious insolubility in
aqueous solutions negatively affects their cellular uptake and
restricts in vivo activity and applications.17 In our previous
study, four cisplatin or transplatin groups were coordinated to

the outer pyridylnitrogens in tetrapyridylporphyrins, indicating
that platinum groups make a significant improvement on
singlet oxygen quantum yields.13,14 They potently inhibited
tumor growth in mice under irradiation for PDT, but not
sufficient for tumor elimination. With the purpose to develop
more efficient photosensitizers available in vivo, oxaliplatin
was chosen as the cytostatic part for the new Pt(II)-porphyrin
photosensitizer, since its DNA binding rate, formation of the
bifunctional adduct, is faster than cisplatin,18 which also can
improve hydrophilicity and eliminate the formation of
aggregates by increasing the electrostatic repulsion from
charged functional groups.19 As far as we know, tetraplatinated
metal-free porphyrins have not yet been investigated for in vivo
PDT.
Where KR and KS are the slopes of the kinetics of DMA and
ABDA disappearance photosensitized by the sample and
standard, respectively. IR and IS are the total light intensities
absorbed by sample and standard, respectively. All
measurements were performed in three times.
Photobiological Assays. Colon26 and Sarcoma180
cell lines were plated in 96-well plates at a density of 5 ×
103 cells/well. The cells were incubated with different
concentrations of 4Pt(dach)ClTPyP, H2TPyP, Pt(dach)Cl2,
and the mixture of H2TPyP and 4 equivalent Pt(dach)Cl2
for 24 h (DMSO < 0.5%). After this period, the complexes
were removed by washing with PBS and the medium was
replaced, which was immediately irradiated by a 50W
LED light (6 J/cm2) or left without irradiation for 30 min.
The plates were kept in the CO2 incubator for 48 h for
further cultivation. Cell viability was estimated by the
standard MTT assay with a microplate reader at an
absorbance of 570 nm.23 All experiments were performed
under dark and light conditions and were replicated for
three times independently.
2. Experimental
Synthesis
5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine (H2TPyP)
and (1R,2R-diaminocyclohexane)(dichloro)platinum(II)
of
4Pt(dach)ClTPyP.
[Pt(dach)Cl2] were synthesized according to the slightly
modified methods as showed in Scheme 1.20 Pt(dach)Cl2
(0.129 mmol, 49 mg) and silver nitrate (0.129 mmol, 22
mg) were dissolved in 2.5 mL of N,N-dimethylformamide
(DMF) and stirred at 60 °C for 24 h. The resulted turbid
solution was filtered to remove white silver chloride. The
light yellow colored filtrate was added to a solution of
H2TPyP (0.0325 mmol, 20 mg) in 2 mL of DMF. After
stirring at 50 °C for 72 h, the mixture was cooled down to
ambient temperature and then precipitated with diethyl
ether. The solid was filtered and further washed with cold
water, cold acetone, chloroform, and diethyl ether. The
solid was dried in vacuum and 4Pt(dach)ClTPyP (dach =
1R,2R-diaminocyclohexane) was obtained as a purple
powder (57 mg, 78%). UV/Vis spectrum: λmax (DMSO,
nm) (ε, 104 M-1 cm-1) 423 (29.6), 516 (1.68), 550 (0.57),
589 (0.54), and 644 (0.22). 1H NMR (400 MHz;
DMSO-d6): δ 9.23 (o-pyridyl, d, 8H), 9.02 (β-pyrrole, s,
8H), 8.51 (m-pyridyl, d, 8H, J = 6.4 Hz), 6.67-5.74 (NH2,
m, 16H), 2.04 (CH, m, 8H), 1.59-1.16 (CH2, m, 32H), 7.95
(CH, DMF), 2.89 (CH3, DMF), 2.73 (CH3, DMF), 2.09
Cellular Uptake. To measure the cellular uptake of
4Pt(dach)ClTPyP, Colon26 cells were treated with 15 μM
4Pt(dach)ClTPyP at 37 °C for 2 h, 6 h and 24 h. Then the
medium was removed, the cells were washed with PBS
solution, harvested, and centrifuged. The pellet was
suspended in a sucrose solution and incubated with buffer
A (10 mM Hepes pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1.0
mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride)
on ice for 15 minutes. After adding 5% NP-40, the
suspension was centrifuged and the supernatant was
collected to get the cytosol. The pellet was re-dissolved in
a sucrose solution and incubated with buffer B (20 mM
Hepes pH 7.9, 0.4 M NaCl, 2 mM EDTA, 1.0 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) on ice
and centrifuged to obtain the pure nuclear extract and
membrane.24 Cellular platinum levels were expressed as
ng of Pt per million cells.
Apoptosis Assay by Flow Cytometry. Colon26 cells
were incubated with 0.5 μM or 10 μM 4Pt(dach)ClTPyP
for 24 h. The medium was changed, irradiated or kept in
dark for 30 min, and continuously incubated for 24 h.
Cells were washed with PBS, and then resuspended in
Annexin V binding buffer. Apoptosis detection kit solution
was added, and the cells were incubated for 15 min in dark.
The cells in different apoptosis stages were analyzed by
flow cytometry.25
Determination of Caspase Activity. After treatment
with 4Pt(dach)ClTPyP for 24 hours, cells were washed
PBS, irradiated for 30 min or kept in dark, and incubated
for different times. Cells were collected and lysed in lysis
buffer (1% Triton X-100, 0.32M sucrose, 5 mM EDTA, 10
mM Tris-HCl, pH 8, 2 mM dithiothione, 2 mM
phenylmethanesulfonyl fluoride) for 20 min at 4 °C
followed by centrifugation (10000 g) for 30 min. The
cytosolic extracts were mixed with 5 μM Ac-DEVD-MCA
(Ac-Asp-Glu-Val-Asp-MCA) in reaction buffer (100 mM
HEPES, 10% sucrose, 5 mM dithiothreitol). Fluorescence
was measured after 3 h (excitation wavelength, 365 nm;
emission wavelength, 460 nm) with a fluorescence
microplate reader.26,27
Animal Model. Animal experiments were conducted
in strict accordance with the Guidelines for the Care and
Use of Laboratory Animals of Kanazawa University. The
animal experimental protocols used were approved by the
Committee on Animal Experimentation of Kanazawa
University (Permit Number: AP-153334). Female BALB/c
(CH3, CH3COCH3); MS (ESI): m/z
=
2115.0
[M-NO3-2Cl+3H]+, 1893.9 [M-2NO3-2(dach)+H]+, 1307.6
[M-4NO3-2{Pt(dach)Cl}]+,
963.5
812.4
[M-4NO3-3{Pt(dach)Cl}]+,
[M-4NO3-3{Pt(dach)Cl}-{(dach)Cl}]+.
Singlet Oxygen Quantum Yields. Singlet oxygen
quantum yields (Φ∆) were determined by photooxidation
of
9,10-dimethylanthracene
(DMA)
and
9,10-anthracenediyl-bis(methylene)dimalonic
acid
(ABDA) via steady-state photolysis. Tetraphenylporphyrin
(TPP) in DMSO ( Φ∆R = 0.52) and Rose Bengal (RB) in
aqueous solution
(
Φ∆R
=
0.76) were used as
references.21,22 DMA solution (1.45 × 10−4 M in DMSO)
or ABDA solution (2 × 10−4 M in aqueous solution) was
mixed with the photosensitizer in a 1-cm quartz cuvette.
The initial absorbance reading of the reaction mixture was
taken at 401 nm or 380 nm, and the cuvette was irradiated.
The kinetics of DMA and ABDA photooxidation were
measured, and the observed rate constants were obtained
by a linear least-squares fit of the semilogarithmic plot of
lnA0/A versus time. The singlet oxygen generated by a
photosensitizer was calculated using the following
equation:
KSIR
ΦS∆ = Φ∆R
KRIS

mice (5 weeks) were purchased and the Colon26 tumor
models were generated by subcutaneous injection of 1×106
cells into the right and left shoulders respectively. The
mice were used for photodynamic therapy and
biodistribution experiments when the tumor volume
reached about 100 mm3.
In Vivo Biodistribution. Three mice per group were
administrated 4Pt(dach)ClTPyP at doses of 8.25 μmol/kg
body weight. The main tissues and blood were collected at
degradation of absorbance and bathochromic shift of the
absorption spectra, indicating that 4Pt(dach)ClTPyP is stable
and illumination cannot evoke the detachment of the peripheral
Pt groups from porphyrin framework. 4Pt(dach)ClTPyP is
soluble in DMSO and water. The hydrophilicity was also
determined by the partition coefficient in an n-octanol/water
system. With a logP value of −1.56 ± 0.24, 4Pt(dach)ClTPyP
displayed high hydrophilicity, which was expected to be
available in vivo.
2,
24,
and
48
h
after
administration.
Tetramethylammonium hydroxide (TMAH) as an aqueous
25% solution was added to each sample and incubated for
24 h at 60 °C. After centrifugation, 0.5 mL of supernatant
was mixed with 4 mL of the solution containing HNO3 1%
and Triton X-100 0.01%.28 Treated samples were analyzed
by monitoring Pt content by ICP-AES.
Photodynamic Therapy. When the tumor sizes of
five mice reached approximately 100 mm3, eight mice
were randomized into 2 groups. Mice in group 1 were
injected into the tail vein with 4Pt(dach)ClTPyP at a drug
dose of 8.25 μmol/kg of body weight. Mice in group 2
were not administrated as control. The tumor of left
shoulder on each mouse in these two groups was irradiated
for 30 min (96 J/cm2) which was divided into three 10 min
periods with a 5 min break between each period, and the
right tumor was kept in dark. The rest of each mouse was
also covered in aluminum foil to avoid organ damage
caused by light exposure. Mice were monitored for tumor
growth and body weight up to 18 days post PDT
treatment.29,30 Tumor size was measured with a digital
caliper and tumor volume was calculated as vol = LW2/2,
where L is the longer diameter and W is the shorter
diameter.
Scheme 1. Synthesis of 4Pt(dach)ClTPyP.
Figure 1. The aggregation behaviors of 4Pt(dach)ClTPyP were
investigated at different concentrations ranging from 0.5 to 10
μM: (a) 4Pt(dach)ClTPyP in DMSO; (b) 4Pt(dach)ClTPyP in
H2O.
Statistical Analysis. Data are expressed as means ±
standard deviations where appropriate. Analyses of in vitro
PDT assay were performed using GraphPad Prism
Software, version 5 for Windows (GraphPad Software,
San Diego, CA, USA). In vivo PDT experiment,
Singlet Oxygen Quantum Yields. Singlet oxygen,
generated by Type II photosensitized reaction, is considered a
major cytotoxic factor responsible for the photodynamic
effect.31-33 Therefore, the PDT efficacy of 4Pt(dach)ClTPyP
was measured by determining the singlet oxygen quantities
under irradiation. The generated singlet oxygen quantities were
given as singlet oxygen quantum yield (Φ∆) values by the
different oxygen quenchers in DMSO and H2O. The results
showed high singlet oxygen quantum yields (Φ∆ = 0.64 ± 0.09
in DMSO and 0.76 ± 0.01 in H2O), while the yield is 0.23 ±
0.05 for H2TPyP in DMSO (not detected in H2O because of
poor solubility), which indicated that introducing oxaliplatin
differences were identified by
a paired Student’s
two-tailed t-test and were considered statistically
significant when p < 0.05.
3. Results and Discussion
Synthesis and Characterization. Scheme 1 illustrates
the synthetic route for formation of 4Pt(dach)ClTPyP. For the
design of 4Pt(dach)ClTPyP, we used H2TPyP as the
photosensitizing unit and the Pt(dach) group as the cytostatic
moiety. H2TPyP and Pt(dach)ClNO3 was synthesized firstly,
and then the product was obtained by conjugation reaction of
these two moieties in 78% yield, which was characterized by
1H NMR (Figure S1-3), ESI mass spectrometry (Figure S4),
UV/Vis spectroscopy (Table S1), and fluorescence
spectroscopy (Figure S5). Upon excitation at 425 nm,
4Pt(dach)ClTPyP manifested a strong emission peak at 647 nm
in DMSO. The UV/Vis spectra of 4Pt(dach)ClTPyP exhibited a
strong Soret band centred at 423 nm resulting from the
increased average electron density of the porphyrin framework
lowering the energy for electron transition, and four Q bands at
516 nm, 550 nm, 589 nm, and 644 nm in DMSO. Strictly
obeying the Beer–Lambert law, this complex did not aggregate
at concentrations from 0.5 μM to 10 μM in DMSO and H2O
(Figure 1). It is meaningful for 4Pt(dach)ClTPyP since
aggregation can greatly decrease the effectiveness of the
photosensitizer in PDT. The photostability of the conjugator
4Pt(dach)ClTPyP has also been evaluated under illumination
for 30 min. As shown in Figure S6, there is no noticeable
1
groups into the peripheral pyridine groups can increase O2
generation by heavy atom effect. Thus, 4Pt(dach)ClTPyP is a
highly efficient singlet oxygen generator and has considerable
potential as a photosensitizer for PDT.
Photobiological Assays in Vitro. The dark cytotoxicity
and photocytotoxicity of the complex towards Colon26 and
Sarcoma180 cell lines were tested using the MTT assay (Table
1 and Table S2). In Table 1, H2TPyP does not show appreciable
antitumor activity under dark or light even at the high
concentration of 400 μM, and the oxaliplatin prodrug,
Pt(dach)Cl2, can efficiently inhibit tumor cell growth with a few
µM to tens of µM, but not photosensitive activity. After
H2TPyP was coordinated to Pt(dach)Cl2, a large increase in
photocytotoxicity towards both cell lines was found for
4Pt(dach)ClTPyP. 4Pt(dach)ClTPyP exhibited low dark
toxicity with IC50 values of 12.50 and 17.85 μM in Colon26
and Sarcoma180 cell lines. Once illuminated, it exhibited

decreased cell viability and IC50 values were determined as
0.17 and 0.25 μM, respectively. Accordingly, peripheral
combination of platinum groups to the porphyrin framework,
generating considerable Φ∆, makes a substantial contribution to
photocytotoxicity, requiring further study to elucidate the
mechanism. Meanwhile, the photocytotoxicity of a mixture of
H2TPyP and 4 equivalents of Pt(dach)Cl2 was also studied as a
control experiment. The IC50 values of this mixture (2.24 and
4.04 μM in Colon26 and Sarcoma180 cell lines) were
significantly larger than those of 4Pt(dach)ClTPyP, completely
proving the success of our synthesis design by combination of
the porphyrin framework and the Pt(II)-based complex.
vivo.25 To verify the mode of cell death caused by
4Pt(dach)ClTPyP-induced PDT, apoptosis assay was
performed by the Annexin V-FITC and PI method, and
cisplatin was used as control (Figure S7). As observed in
Figure 3, the untreated cells were Annexin V and PI negative
under irradiation or dark, indicating that the cell membrane was
intact and the cells did not undergo apoptosis. After treatment
with 4Pt(dach)ClTPyP with light, the percentage of Colon26
cells at the apoptotic stage was 61.24%. This observation
clearly indicated that Colon26 cells underwent an apoptotic
pathway when PDT-treated with 4Pt(dach)ClTPyP.
Table 1. IC50 values (μM) towards Colon26 cell line
Colon26a
Compound
PIb
In dark
With light
4Pt(dach)ClTPyP
H2TPyP+4Pt(dach)Cl2
Pt(dach)Cl2
12.50±5.98 0.17±0.09
1.87±0.71 2.24±0.08
1.37±0.53 3.40±1.93
74
0.8
0.4
—
1
H2TPyP
> 100
> 100
cisplatin
3.62±1.00 3.25±0.91
a
All compounds were examined in a set of experiments
repeated three times independently.
b PI: phototoxic index (PI = IC50 in the dark/IC50 in light).
Subcellular Localization. In order to investigate cell
localization of the promising 4Pt(dach)ClTPyP complex,
cellular uptake studies were carried out. Colon26 cells were
incubated with 4Pt(dach)ClTPyP for 2, 6, and 24 h, and the
platinum content was determined in the cytosol, nucleus, and
membrane fractions using ICP-AES. The data, reported as ng
of platinum per million cells, are shown in Figure 2a. From the
results, 4Pt(dach)ClTPyP exhibited fast cellular uptake and
reached a maximum concentration at 6 h. A large portion was
detected in the cell membrane fraction containing mitochondria
where heme is synthesized.34 The complex was distributed in
the nucleus, which may result from platinum groups localizing
in cell nuclei and insertion into DNA.16,35 4Pt(dach)ClTPyP
also displayed an unignorable distribution in cytosol, which
may cause mitochondrial damage that affects the energy
production of cells, activating the caspase-3- and 9-mediated
apoptotic pathway.36
Figure 3. Flow cytometric analysis of Colon26 cells with or
without 4Pt(dach)ClTPyP showing % apoptotic cell death.
Caspase-3 Induced Apoptosis. Porphyrin-based
photosensitizers for PDT may be capable of inducing rapid
release of cytochrome c and initiating an apoptotic cascade.
After releasing mitochondrial cytochrome c into the cytosol,
caspase-3 can be activated triggering apoptotic cell death.36,37
To identify whether caspase-3 is involved in PDT induced
apoptosis, Ac-DEVD-MCA,
a
caspase-3-like proteinase
substrate, was employed for estimating the catalytic activity of
caspase-3 (Figure 2b). 4Pt(dach)ClTPyP-treated cells could not
be significantly triggered to active caspase-3 under dark
conditions. Whereas, caspase-3 activity increased under light
irradiation, indicating that 4Pt(dach)ClTPyP can lead to cell
apoptosis involving caspase-3 with light.
In vivo Biodistribution. The in vivo pharmacokinetic
activity of 4Pt(dach)ClTPyP after tail-vein administration was
investigated in Colon26 tumor-bearing mice and
Pt-concentration in each tissue is given in Figure 4. After
intravenous administration, the complex accumulated in the
spleen, kidney, liver, and lung during the early stage. After 24 h
post-injection, the Pt concentration dramatically reduced in
blood. It has been argued that porphyrin-based photosensitizers
can selectively accumulate in tumors, since cancer cells
overexpress low density lipoproteins which would play an
important role in transportation of porphyrins to tumor
cells.38,39 Using blood and muscle as two reference tissues, the
concentration ratios of tumor-to-blood and tumor-to-muscle
increased with time, reaching 2.9 and 31.5 at 48 h respectively.
Whereas, the preferential uptake of 4Pt(dach)ClTPyP was not
found when compared with other organs. We hypothesized that
some Pt groups may be detached from the porphyrin
framework.
Figure 2. (a) Cellular uptake following 4Pt(dach)ClTPyP
treatment (15 μM for 2 h, 6 h, and 24 h) in Colon26 cell line;
(b) Effects of 4Pt(dach)ClTPyP-induced caspase-3 activation in
Colon26 cells. Colon26 cells were incubated with 0.1 μM
4Pt(dach)ClTPyP for 24 h with and without light irradiation,
and Ac-DEVD-MCA was used as the substrate for caspase-3.
The caspase-3 activity of control cells was set to 100% and the
relative changes in the activity under light are shown.
Apoptosis Assay by Flow Cytometry. It is suggested
that PDT can trigger apoptosis and necrosis in vitro and in

Figure 6. In vivo PDT evaluation of 4Pt(dach)ClTPyP in
Colon26 tumor-bearing mice. (a) Tumor growth curves of
group 1 (4Pt(dach)ClTPyP, n = 4) and group 2 (Control, n = 4).
The tumor volumes were normalized to their initial sizes. (b)
Relative weight of group 1 and group 2. The error bars
represent the standard deviations of 4 mice per group. *, p <
0.05; **, p < 0.01, ***, p < 0.001.
Figure 4. Pt-concentration in each tissue at 2, 24, and 48 h after
intravenous injection of 4Pt(dach)ClTPyP (n = 3; Bld = blood;
Spl = spleen; Pan = pancreas; Sto = stomach; L-Int = large
intestine; S-Int = small intestine; Kid = kidney; Liv = liver; Hrt
= heart; Lng = lung; Mus = muscle; Brn = brain; Tum =
tumor).
cisplatin-porphyrin
complexes
we
reported
before,
In vivo PDT on Subcutaneous Tumor-Bearing Mice.
To evaluate the therapeutic effect of 4Pt(dach)ClTPyP in vivo,
further experiments were conducted towards Colon26
tumor-bearing mice by monitoring the tumor volume. Mice in
group 1 were intravenously injected with 4Pt(dach)ClTPyP and
mice in group 2 were not administrated as control. Each mouse
in these two groups underwent irradiation on a tumor located
on the left shoulder and no irradiation on the right shoulder.
Considering the possibility of low accumulation in tumors and
metabolic degradation, light irradiation was performed after 2 h
administration to obtain enhanced PDT effect due to the
maximum blood concentration of the photosensitizer. To assess
the side effects, the body weight was also measured. As
indicated in Figure 5 and Figure 6a, the tumor on both
shoulders of group 2 and on the right shoulder of group 1 were
not inhibited and showed similar growth speeds. When
comparing the tumor volumes of group 2, there was no
significant difference between the left shoulder and right
shoulder, suggesting that light irradiation only does not affect
tumor growth. No obvious difference was found between the
tumor on right shoulders of both groups, hence, dark
cytotoxicity of 4Pt(dach)ClTPyP is insignificant; however, the
4Pt(dach)ClTPyP is more hydrophobic and its superior PDT
effect could be attributed to relatively high cellular uptake
(logP value of 4cisplatin-porphyrin: −1.77 ± 0.08). Additionally,
in the treated group, body weight measurements showed that
the mice had no appreciable weight loss (less than 6%, Figure
6b). Skin damage was detected after administration, but
recovered with time (Figure 5). All of the results indicate that
4Pt(dach)ClTPyP is capable of tumor removal in Colon26
tumor-bearing mice and could be an effective agent in future
clinical applications for PDT.
4. Conclusion
In summary, to obtain a potential photosensitizing agent
with high antitumor effect, a porphyrin framework as a
photosensitive group and a Pt (II)-based drug were combined
and characterized. It is hypothesized that the positive
charge-rich regions of this complex prohibit formation of
aggregates by electrostatic repulsion and facilitate
metal-to-ligand charge transition to increase singlet oxygen
quantum. The synthesized complex, 4Pt(dach)ClTPyP, showed
negligible dark toxicity and excellent photocytotoxic properties.
The reasons for cell death can be attributed to high singlet
oxygen quantum, internalization into nucleus, and an apoptosis
pathway. In vivo PDT efficacy of this complex was conducted
using Colon26 tumor-bearing mice via tail vein injection, and
this complex exhibited splendid cancer killing efficacy over
two weeks in an in vivo PDT assay. Based on these
experimental results, 4Pt(dach)ClTPyP is a highly promising
anticancer agent for PDT.
left tumor in group
1 disappeared on day 4 after
4Pt(dach)ClTPyP-mediated PDT and remained absent for 18
days (P < 0.05 vs. the right tumor volume). Therefore, this
complex has been demonstrated to completely killed tumor
cells, rather than inhibiting tumor growth. Compared with the
The author (AO) gratefully acknowledges The Ministry of
Education, Culture, Sports, Science, and Technology in Japan
(25288027) for support of the part of the work.
Supporting Information
An additional experimental results including H NMR spectra,
1
ESIMS spectrum, emission spectra, photostability profiles,
photophysical and photochemical data and photobiological data.
This material is available on http://dx.doi.org/10.1246/bcsj. .
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Figure 5. BALB/c mice bearing Colon26 cancer cells before
photoirradiation (1a and 2a), 6 days (1b and 2b), and 18 days
(3a and 3a) after PDT treatment of 4Pt(dach)ClTPyP. The
mouse in 1a, 1b, and 1c was in group 1, and the mouse in 2a,
2b, and 2c was in group 2.
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