Enhanced in vitro photocytotoxicity of water-soluble dendritic pheophorbide-a

Article abstract of DOI:10.1142/S1088424615500546

Photosensitizers can produce highly reactive singlet oxygen with exposure to visible light and are used in photodynamic therapy to treat a variety of tumors. We report on the synthesis of triethylene glycol dendron-conjugated pheophorbide-a 5, a novel pho

Full text of DOI:10.1142/S1088424615500546

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2015; 19: 830–837
DOI: 10.1142/S1088424615500546
Published at http://www.worldscinet.com/jpp/
Enhanced in vitro photocytotoxicity of water-soluble
dendritic pheophorbide-a
Eun Ji Leea, Hyoung Jun Konga, Young-Jin Kimb, Jong S. Parkc
and Myung-Seok Choi*a◊
a Department of Materials Chemistry and Engineering, Konkuk University, Seoul 143-701, Korea
b Department of Biomedical Engineering, Catholic University of Daegu, Gyeongsan 712-702, Korea
c Department of Organic Material and Polymer Engineering, Dong-A University, Busan 604-714, Korea
Received 2 February 2015
Accepted 28 February 2015
ABSTRACT: Photosensitizers can produce highly reactive singlet oxygen with exposure to visible
light and are used in photodynamic therapy to treat a variety of tumors. We report on the synthesis
of triethylene glycol dendron-conjugated pheophorbide-a 5, a novel photosensitizer. The characteristic
absorption bands (Soret and Q-bands, lmax = 405 and 670 nm, respectively) of 5 were appeared clearly
in aqueous solution, due to the improved water-solubility of the dendron moiety. The value of singlet
oxygen quantum yield of 5 (FD = 0.22) was higher than free pheophorbide-a (FD = 0.17) as reference in
aqueous solution. Compound 5 also exhibited an enhanced in vitro phototoxicity than pheophorbide-a
(PhA) in the concentration range of 1.0–5.0 mg/mL: cell viability in cells treated with 5 was reduced by
~20%, indicating a cell death rate of ~80%, while PhA treatment resulted in a cell death rate of only
about 10%. These results indicate that 5 will likely be more efficient in PDT applications. Compared
with free PhA, compound 5 showed highly enhanced singlet oxygen generation ability and in vitro
photocytotoxicity.
KEYWORDS: pheophorbide-a, photosensitizer, photodynamic therapy, singlet oxygen,
photocytotoxicity.
tetrapyrrolic compounds, including porphyrins [4],
INTRODUCTION
chlorins [5], bacteriochlorins [6], and phthalocyanines
Photodynamic therapy (PDT) is a photochemical
[7], are used widely as photosensitizers for both clinical
treatment in which biological targets are destroyed with
and experimental applications of PDT [8]. The first PDT
reactive oxygen species, primarily superoxide ions (O2-)
agent used in clinical practice was “Photofrin,” a mixture
and singlet oxygen (1O2), generated by a light-driven
of oligomers formed by ether and ester linkages of up
photosensitizing process [1]. Upon absorption of light,
to eight porphyrin units [9]. Porphyrin-based PS agents
a photosensitizer (PS) generates excited triplet states via
have, however, been shown to have some undesirable
properties including poor absorption in the PDT optical
window (>ꢀ630 nm) and relatively long retention times
in biological systems. To overcome these limitations,
excited singlet states, which readily transfer excitation
energy to the ground state of molecular oxygen (3O2)
to produce singlet oxygen (1O2), which, in turn, readily
reacts with biological tissues [2]. The inhibitory effects
chlorine-based PS agents, such as m-tetrahydroxyphenyl
of PDT can be achieved by inducing apoptosis and
chlorin and mono-aspartyl chlorin e6, have been
systemic antitumor immunity through the activation
developed in the past decade [10, 11]. The chlorin
of neutrophils and natural killer cells [3]. Various
derivative pheophorbide-a (PhA) is a promising PS, and
has received attention because of its potential application
in PDT [12]. Most conventional PS-conjugated drug
◊ SPP full member in good standing
delivery systems readily aggregate in aqueous media due
to strong p–p interactions among PS molecules, resulting
*Correspondence to: Myung-Seok Choi, email: mchoi@konkuk.
ac.kr
Copyright © 2015 World Scientific Publishing Company

ENHANCED IN VITRO PHOTOCYTOTOXICITY OF WATER-SOLUBLE DENDRITIC PHEOPHORBIDE-a
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1
in the suppression of O2 generation by self-quenching.
by alkali conditions. Compound 2 was prepared from
the coupling reaction of 3,5-dihydroxybenzyl alcohol
with tosylated triethylene glycol monomethyl (1).
Esterfication of 2 with 3,5-dihydroxybenzyl alcohol
using DEAD/TPP was followed by the terminal ester-
reduction to produce 4. Finally, the synthesis of target
compound 5 was carried out with 4 and PhA under
DCC/DMAP coupling conditions. Compound 5 was
purified by recycling GPC and characterized structurally
Accordingly, PhA is used to chemically/physically bind
to solubilizing PS-carriers, including human serum
albumin (HSA) [13], Au nanoparticles [14], pullulans
[15], PEGylated polymers [16], polymeric micelles
[17], and PMMA dendrimers [18] for high therapeutic
efficacy. Dendrimers in particular have been investigated
widely as carriers due to the relatively facile size-
tunability and surface-functionality they exhibit [19].
A dendrimer-encapsulated porphyrin sensitizer was, for
example, shown to have strong photodynamic efficacy
and singlet oxygen generation [20]. It is also well known
that ethylene glycol moieties inhibit aggregate formation
and enhance cytotoxicity and tumor-targeting efficacy
[21]. It was thus of interest to incorporate hydrophilic
dendron units for efficient photodynamic activity. In
this study, a dendritic PhA derivative with tri(ethylene
glycol) dendron (TEG-dendron) was designed and
synthesized, which showed relatively enhanced in vitro
photocytotoxicity.
1
by UV-vis absorption, fluorescence emission, HNMR
spectroscopy, and MALDI-TOF mass spectrometry.
Size-exclusion chromatography (SEC) of 5 and PhA
produced sharp elution peaks at retention time (tR) =
16.8 and 22.3 min, respectively, with minor peaks from
column impurities (Fig. S1). The structure of 5 was
1
confirmed by H NMR spectrum in CDCl3, where the
peripheral methyl group of TEG-dendron appeared at
3.36 ppm as a single peak, while the other TEG-dendron
protons appeared over 3.54–4.14 ppm as multiple peaks.
The protons from the PhA moiety appeared at 7.63, 6.91,
5.31, 2.18, 1.58 and 1.27 ppm as a single or multiple
peaks (Fig. S2a). As can be seen from the MALDI-TOF
mass spectrum of 5 (Fig. S2b), a most intense peak at
m/z = 1543.82 was assigned to the molecular ion [M +
H]+ (calculated value: m/z = 1543.76) and a minor peak
at m/z = 1566.79 was observed for the sodium-coupled
molecular ion [M + Na]+ (calculated value: m/z =
1565.75) where the sodium ion is thought to come
from the brine used in the washing process. A partially
defective structure (fragmentation) at m/z = 1033.54 was
also observed with low intensity.
RESULTS AND DISCUSSION
Synthesis and structural characterization
As shown in Scheme 1, the overall synthesis of 5 was
carried out according to a previously described method
with slight modifications [25]. Briefly, compound 1
was produced through tosylation of triethylene glycol
monomethyl with p-toluenesulfonyl chloride, promoted
O
O
O
O
HO
HO
O
O
O
O
O
CH2OH
CO2CH3
O
O
O
O
O
O
O
O
HO
HO
O
O
O
OTs
CH2OH
X
a
c
b
O
O
O
O
1
O
2
O
O
O
3
4
X = CO2CH3
= CH2OH
O
O
O
O
CH2
CH2
H3C
NH
H3C
NH
O
N
N
N
N
CH3
CH3
H3C
H3C
O
O
O
O
HN
HN
O
O
HO
H3C
H3C
O
O
d
O
O
O
O
O
O
O
CH3
CH3
H3CO
H3CO
O
O
ref. pheophorbide-a (PhA)
O
5
O
O
O
Scheme 1. Synthetic routes of compound 5: (a) 18-crown-6, K2CO3, THF; (b) diethyl azodicarboxylate (DEAD, 40% in toluene),
PPh3, THF; (c) LiAlH4, THF; (d) pheophorbide-a, DCC, DMAP, THF
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Absorption and emission properties
during circulation in the blood. The fluorescence of 5
may be recovered by decomposition of the colloidal
particles in intracellular environment.
The absorption and emission spectra of 5 with some
references are shown in Fig. 1, where the concentration
of all samples was adjusted to 0.28 mM. As shown in
Fig. 1a, the UV-vis absorption spectrum of 5 in THF
showed an intense and broad Soret band at lmax = 412 nm
together with relatively weak Q-Bands at lmax = 507,
536, 611 and 667 nm. These absorption characteristics
of 5 correspond closely with those of a THF solution
of PhA, indicating that there is a negligible/weak
electronic interaction between the PhA moiety and
the TEG-dendron moiety in 5. In PBS buffer, free
PhA does not show characteristic absorptions because
it is not soluble at all. On the other hand, compound
5 showed the typical absorption features (Soret and
Q-bands, lmax = 405 and 670 nm, respectively) in the
visible region due to the improved solubility of the
TEG-dendron moiety, which is considerably different
to a THF solution. The Soret band of 5 was significantly
decreased and broadened compared with that of 5
in THF. These results indicate that stable colloidal
aggregations of 5 were formed in aqueous medium due
to strong hydrophobic and p–p interactions among PhA
moieties, where the particle size was determined to be
~200 nm by dynamic light scattering measurements
(Fig. S3). Particle sizes of >200 nm can be problematic
in PDT applications due to the resulting insufficient
circulation time in the bloodstream [26]. As shown in
Fig. 1b, the emission characteristic of 5 (lmax = 671 nm;
Singlet oxygen generation
1
The O2 generation ability of 5 was determined by
measuring the fluorescence intensity of 1,3-diphenyliso-
benzofuran (DPBF) with time (Fig. 2). In this process,
photobleaching of the DPBF can be used to monitor
singlet oxygen generation where DPBF reacts readily
1
with O2, causing a decrease in fluorescence intensity
of DPBF in the range of 420–600 nm. In DMF solution
(Fig. 2a), the DPBF fluorescence intensity was slightly
decreased, by 10%, at an early irradiation time (120 s).
At the middle stage of the irradiation (240–360 s),
fluorescence was quenched by 60% of the initial intensity.
Finally, the fluorescence intensity of DPBF was largely
quenched, by 95%, at the final stage of the irradiation
time (600 s), as compared with no irradiation. In contrast,
a solution of 5 in PBS buffer (Fig. 2b) showed a relatively
smaller decrease (~20%) in DPBF fluorescence with
irradiation time (120–720 s) because particles that do
not facilitate efficient triplet-triplet energy transfer from
1
5 to oxygen molecules (3O2) to give O2 are formed.
These results correlate with the change in fluorescence
intensity measured for 5, as described above. A solution
of PhA in DMF and PBS buffer, as a reference, showed
slightly higher and lower photobleaching behaviors in
DPBF fluorescence compared to those of 5 (Fig. 2c),
respectively. Thus, compound 5 was found to have an
efficient ability to generate singlet oxygen, similar to
PhA with improved water solubility. The singlet oxygen
quantum yield (SOQ) was evaluated according to a
previous report [27]. The value of SOQ of 5 (FD = 0.49)
was slightly lower than PhA (FD = 0.52) in DMF solution.
l
ext = 412 nm) in THF at room temperature were found
to be very similar to those measured for PhA (lmax
=
671 nm). The fluorescence intensity of 5 in PBS was
largely reduced, by 97%, compared with the intensity
of 5 in THF, because fluorescence quenching occurs
readily by collisions between molecular excited states in
colloidal particles, which would lead to low cytotoxicity
Fig. 1. (a) Absorption and (b) emission spectra of 5 and PhA in THF or PBS
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ENHANCED IN VITRO PHOTOCYTOTOXICITY OF WATER-SOLUBLE DENDRITIC PHEOPHORBIDE-a
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Fig. 3. Cell viability assessment of MCT-7 cells with 5 and PhA
(a) without light irradiation and (b) with light irradiation
In vitro phototoxicity
The in vitro phototoxicity of 5 in breast cancer cells
(MCF-7) was examined using the MTT assay, which
is a colorimetric assay to assess cell viability. In the
assay, a yellow tetrazole MTT is reduced to the optically
detectable purple formazan by living cells [28]. Upon
light irradiation with 100 mW/cm2 at 670 nm, 5 displayed
higher phototoxicity than PhA in the concentration range
of 1.0–5.0 mg/mL: cell viability in cells treated with 5 was
reduced by ~20%, indicating a cell death rate of about
80%, while PhA treatment resulted in a cell death rate
of only ~10% (Fig. 3). In contrast, the dark cytotoxicity
(without light treatment) was significantly lower, showing
a cell death rate of ~10% for PhA and ~20% for 5. There
was no significant difference in the dark cytotoxicity
between 5 and PhA because an acceptable cell viability
above 80% was obtained in the concentration range of
1.0–5.0 mg/mL. The result of MTT assay indicated that 5
is more efficient in photodynamic theraphy than PhA due
to improved water-solubility.
Fig. 2. Photobleaching of DPBF fluorescence (lext = 360 nm)
upon excitation of 5 with 670 nm laser for 0, 120, 240, 360,
480 and 600 s (a) in DMF (FD = 0.49) (b) or in PBS buffer
(FD = 0.22) at room temperature. (c) Plot of change in DPBF
fluorescence intensity of 5 vs. PhA in DMF and PBS
However, 5 (FD = 0.22) in PBS showed an increase in
SOQ value, by ~30% vs. that of PhA (FD = 0.17). These
results indicate that 5 will likely be more efficient in PDT
applications.
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E.J. LEE ET AL.
Fig. 4. Apoptotic cell death study in MCF-7 cells treated with (a) PhA and (b) 5 using Annexin V-FITC
The phototoxicity-induced apoptosis in cells treated
with 5 was assessed by an Annexin V-FITC study.
Apoptotic cells lose the asymmetry of membrane
phospholipids,resultinginexposureofphosphatidylserine
on the outer leaflet of the plasma membrane. Cells were
thus stained with Annexin V-FITC, which binds with
phosphatidylserine and gives a green emission at the
outer membrane [29]. It is well known that PhA can
cause apoptotic cell death. As shown in Fig. 6, a stronger
fluorescent signal, indicative of apoptotic cells, was
detected in irradiated MCF-7 cells treated with 5 (Fig. 4b)
than in irradiated cells treated with PhA (Fig. 4a).
Characterization
Basic contamination in the reaction mixture was
removed by column chromatography using silica gel
60 (Merck, 0.063–0.200 mm). Further purification was
carried out on a recycling preparative HPLC (Japan
Analytical Industry model LC-9201) equipped with
column set consisting of JAIGEL-2H and JAIGEL-2.5H
with THF as the eluent at a flow rate of 3.5 mL.min-1.
Analytical size-exclusion chromatography (SEC) was
performed on a Young Lin Instrument (model YL 9100)
with DMF as the eluent. UV-vis absorption spectra were
recorded on a JASCO V-670 spectrometer and steady-
state fluorescence spectra were measured with a Perkin
1
Elmer LS 55 fluorescence spectrometer. H NMR data
were recorded on a Mercury Plus 300 MHz spectrometer
at ambient temperature using an internal deuterium lock.
MALDI-TOF mass spectrometry was performed on a
Voyager-DETM STR Biospectrometry Workstation,
equipped with a 337 nm N2 laser producing 3 ns pulses
at a repetition rate to 20 Hz in reflector mode. Particle
size and distribution were measured by dynamic light
scattering (DLS) using a K-ONE model scatteroscope.
EXPERIMENTAL
Materials
4-Dimethyaminopyridine (DMAP), 3.5-dihydroxy-
benzyl alcohol, and methyl-3.5-dihydroxybenzoate were
purchased from Tokyo Chemical Industry. 1,3-Dicyclo-
hexylcarbodiimide (DCC), diethyl azodicarboxylate
(DEAD, 40% toluene solution), diphenylisobenzofuran
(DPBF), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT), and Annexin V-FITC were
purchased from Sigma-Aldrich Co., (St. Louis, MO,
USA).Pheophorbidea(PhA)waspurchasedfromFrontier
Scientific, Inc., (Salt Lake City, UT, USA). Human breast
cancer cell line (MCF-7) was received from the Korean
Cell Line Bank (KCLB, Korea). RPMI-1640, fetal
bovine serum (FBS) and penicillin-streptomycin were
obtained from Gibco BRL (USA). Annexin V-fluorescent
isothiocyanate (FITC) fluorescence microscopy kit was
from BD Biosciences (USA). All chemicals were used
without further purification. Tosylated triethylene glycol
monomethyl ether (1) was prepared according to a
previously described method [22]. Deionized water was
purified using a U Pure Power deionization system.
Synthesis
Compound 1. A solution of NaOH (14.3 g, 0.357 mol)
in water (62 mL) was mixed with a solution of triethylene
glycol monoethyl ether (50.0 g, 0.243 mol) in dry THF
(62 mL), and then a solution of 4-toluenesulfonyl chloride
(55.5 g, 0.291 mol) in dry THF (62 mL) was slowly added
drop-wise. After stirring for 18 h at room temperature,
the reaction mixture was poured into water and extracted
with CH2Cl2 (3 × 50 mL). Collected organic layers were
washed with water and brine, and dried with MgSO4.
The solvent was evaporated and the residue was purified
by column chromatography on silica gel using a solvent
mixtureofCH2Cl2/ethylacetate(9:1)aseluent.Thesecond
band was collected and evaporated to dryness to give a
1
colorless oil (yield 74%). H NMR (CDCl3, 300 MHz):
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ENHANCED IN VITRO PHOTOCYTOTOXICITY OF WATER-SOLUBLE DENDRITIC PHEOPHORBIDE-a
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d, ppm 7.80 (d, J = 4.1 Hz, 2H), 7.35 (t, J = 0.3 Hz, 2H),
4.95 (s, 4H), 4.14–4.09 (m, 12H), 3.87–3.83 (m, 9H),
3.76–3.65 (m, 24H), 3.57–3.54 (m, 10H), 3.36 (s, 12H),
2.19–2.17 (m, 16H), 1.58–1.57 (m, 3H), 1.29–1.25 (m,
3H). MS (MALDI-TOF): m/z 1543.82 (cacld. for [M +
H]+ 1543.76).
4.16 (t, J = 0.6 Hz, 2H), 3.69–3.53 (m, 10H), 3.37 (s, 3H).
Compound 2. A mixture of 3,5-dihydroxybenzyl-
alcohol (7.7 g, 0.055 mol), K2CO3 (76 g, 0.55 mol),
18-crown-6 (5.8 g, 0.022 mol), and 1 (35 g, 0.11 mol)
were added to dry THF (550 mL), and the solution was
refluxed for 12 h. The reaction mixture was concentrated
and worked up by adding an equal volume of CH2Cl2
and water. Collected organic layers were washed with
water and brine, and dried with MgSO4. The solvent
was evaporated and the residue was purified by column
chromatography on silica gel using a solvent mixture
of CH2Cl2/methanol (10:1) as the eluent. The second
band was collected and evaporated to dryness to give a
colorless oil (yield 72%). 1H NMR (300 MHz, CDCl3): d,
ppm 6.53 (d, J = 2.3 Hz, 2H), 6.32 (d, J = 2.3, 1H), 4.52
(s, 2H), 4.18–3.47 (m, 12H), 3.31 (s, 6H), 1.98 (s, 1H).
Compound 3. A 40% toluene solution (11.56 mL) with
diethyl azodicarboxylate (4.43 g, 25.43 mmol) was added
to a THF solution (150 mL) of 3,5-dihydroxybenzoate
(1.95 g, 11.56 mmol), 2 (10 g, 23.12 mmol) and triphenyl
phosphine (6.67 g, 25.43 mol). The solution was stirred
at room temperature for 24 h. The work up was carried
out in a manner similar to that described above for 2. The
crude product was isolated by column chromatography
on silica gel using a solvent mixture of ethyl acetate/
methanol (10:1) as the eluent. The fourth band was
collected and evaporated to dryness to give a colorless oil
Evaluation of singlet oxygen generation
1,4-Diphenyl-2,3-benzofuran (DPBF) was used as
singlet oxygen (1O2) chemical quencher to evaluate the
capability of 5 to generate 1O2 for PDT applications [23].
To prepare solution samples, a DMF solution of 5 (0.97
mM) containing DPBF (20 mM) was 1000-fold diluted
with DMF or PBS buffer (pH 7.4). Sample solutions in
DMF and PBS were continually irradiated by a 670 nm
laser at room temperature while the DPBF fluorescence
emission(lext =360nm)wasrecordedwithtime(0–600s).
The singlet oxygen quantum (SOQ) yield was determined
using the equation described in a previous report [15].
Cell culture and incubation conditions
A human breast cancer cell line (MCF-7) was used for
all cell culture experiments. MCF-7 cells were cultured
in RPMI-1640 medium containing 10% fetal bovine
serum (FBS) and 1% penicillin-streptomycin. Cells were
incubated at 37°C in a humidified 5% CO2 atmosphere.
When the cells reached 80% confluence, they were
harvested by 0.25% trypsin-EDTA and seeded into new
tissue culture plates for subculture. Compound 5 and free
PhA were dispersed in serum-free medium. Irradiated
untreated cells and untreated cells kept in the dark were
used as controls.
1
(yield 70%). H NMR (300 MHz, CDCl3): d, ppm 7.26
(s, 2H), 6.76 (s, 1H), 6.59 (d, J = 2.3 Hz, 4H), 6.46 (d,
J = 2.3 Hz, 2H), 4.99 (s, 4H), 3.91 (s, 3H), 4.13–3.54 (m,
48H), 3.37 (s, 12H).
Compound 4. A solution of 3 (4.2 g, 4.21 mmol)
dispersed in THF (150 mL) was added to a THF solution
(100 mL) of LiAlH4 (0.24 g, 6.32 mol). The solution was
stirred at 4°C for 1 h. The workup was carried out in a
manner similar to that described above for 2. The crude
product was isolated by column chromatography on silica
gel using a solvent mixture of ethyl acetate/methanol
(10:1) as the eluent. The first band was collected and
evaporated to dryness to give a colorless oil (yield 61%).
1H NMR (300 MHz, CDCl3): d, ppm 6.59 (d, J = 2.3 Hz,
2H), 6.58 (d, J = 2.3 Hz, 4H), 6.50 (s, 1H), 6.43 (d, J =
2.3 Hz, 2H), 4.96 (s, 4H), 4.62 (s, 2H), 4.13–3.54 (m,
48H), 3.37 (s, 12H).
Cell phototoxicity assay
To determine cell viability under dark conditions,
MCF-7 cells (1 × 104 cells/well) were seeded in 96-
well plates and incubated for 24 h at 37°C. After cell
stabilization, the culture medium was replaced with
200 mL serum-free culture medium containing 5 or
PhA (0–5 mg/mL), followed by incubation for 4 h. The
cells were then washed twice with serum-free medium
and cell viability was evaluated after 24 h using the
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) assay. To determine in vitro phototoxicity
of 5 and PhA after laser irradiation, MCF-7 cells (1 × 104
cells/well) were seeded in 96-well plates and incubated
for 24 h at 37°C. These cells were then treated with
serum-free medium containing 5 or free PhA (0–5 mg/
mL). After a 4-h incubation, the cells were washed twice
with serum-free medium and irradiated with a 670 nm
diode laser (100 mW/cm2) for 3 min. To avoid any
photothermal-induced cell damage, the irradiation time
was limited to below 9 min [24]. The cell viability of
irradiated cells was evaluated by the MTT assay after
24 h incubation using a microplate reader (OPSYS-MR,
Dynex Technology Inc., USA). The mechanism of this
Compound 5. PhA (0.2 g, 0.34 mmol) was added
to a THF solution (100 mL) of 4 (0.39 g, 0.405 mmol),
DCC (0.1 g, 0.51 mmol) and DMAP (0.06 g, 0.51 mol).
The solution was stirred at room temperature for 24 h.
The workup was carried out in a manner similar to that
described above. The crude product was isolated by
column chromatography on silica gel using a solvent
mixture of ethyl acetate/methanol (10:1) as the eluent.
The second band was collected and evaporated to dryness
1
to give a greenish oil (yield 24%). H NMR (300 MHz,
CDCl3): d, ppm 7.63 (s, 1H), 6.91 (s, 1H), 6.59–6.58
(m, 8H), 6.44–6.42 (m, 2H), 5.31 (s, 1H), 5.04 (s, 4H),
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Cellular apoptosis was visualized using an Annexin
V-FITC fluorescence microscopy kit. MCF-7 cells (1 ×
104 cells/well) were seeded on 8-well chamber slides for
24 h before being treated with 5 or free PhA (0–5 mg/mL)
for 4 h. Cells were then rinsed twice with Dulbecco’s
phosphate-buffered saline (DPBS) and illuminated with
a 670 nm diode laser (100 mW/cm2) for 3 min. The cells
were stained with l mL Annexin V-FITC (10% w/w) for
15 min at room temperature, afterwhich they werewashed
twice with DPBS and fixed using 4% paraformaldehyde
in DPBS for 30 min. A cover slip was mounted on a
microscope slide with a drop of anti-fade mounting
solution to reduce fluorescence photobleaching. Cells
were observed with an inverted fluorescence microscope
(Eclipse TS100, Nikon, Japan) using a filter set for FITC
visualization.
CONCLUSION
In this study, a novel photosensitizer 5 with improved
water solubility was developed. Light irradiation showed
that 5 exhibited efficient O2 generation, and was found
1
to form particles of a suitable size (<ꢀ200 nm in PBS)
for PDT applications. As determined by an MTT cell
viability assay, 5 exhibits a higher phototoxicity than PhA
at concentrations of 1.0–5.0 mg/mL, while no significant
difference was measured in the dark toxicities of the drugs.
AnAnnexinV-FITC assessment clearly demonstrated that
5 was capable of inducing more apoptotic cell death than
PhA. Further work is in progress to optimize 5 towards
reducing in vivo cytotoxicity for clinical applications.
Acknowledgements
This research was supported by a grant from Ministry
of Trade, Industry & Energy, Republic of Korea (Nos.
10047756 and 10047953) and the Small & Medium Busi-
ness Administration, Republic of Korea (No. S2045034).
Supporting information
Figures S1–S3 are given in the supplementary material.
This material is available free of charge via the Internet at
http://www.worldscinet.com/jpp/jpp.shtml.
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