In vitro phototoxicity and dark-toxicity of a novel synthesized pyropheophorbide-a-paclitaxel conjugate against cancer cell lines

Article abstract of DOI:10.1142/S1088424612500757

Synthesis of pyropheophorbide-a-paclitaxel (PPa-PTX) conjugate was performed in high yield with the aim of searching for an optimal agent for cancer treatment. After synthesis, the conjugate was confirmed to be linked through an ester bond at the 2′ position of the paclitaxel moiety using multi-nuclear magnetic resonance spectroscopy. Phototoxicity of PPa and PPa-PTX conjugate, as well as PTX, was evaluated with three human cancer cell lines (HeLa, CaSki and TC-1). The new conjugate at 0.010.06 μM displayed 2040% higher phototoxicity in HeLa and CaSki cell lines than free PPa and PTX. Furthermore, cellular uptake of these bio-molecules was examined by confocal laser scanning microscopy. Although PPa-PTX showed a delayed uptake compared to PPa, it penetrated completely into cells within 24 h incubation.

Full text of DOI:10.1142/S1088424612500757

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 1006–1014
DOI: 10.1142/S1088424612500757
Published at http://www.worldscinet.com/jpp/
In vitro phototoxicity and dark-toxicity of a novel synthesized
pyropheophorbide-a-paclitaxel conjugate against cancer cell
lines
Gantumur Battogtokha, Hai-Bo Liua, Su-Mi Baea, Pankaj K. Chaturvedia,
Yong-Wan Kima, In-Wook Kima and Woong Shick Ahn*a,b
a Cancer Research Institute, Catholic Research Institute of Medical Science, The Catholic University of Korea, 505
Banpo-dong, Seocho-gu, Seoul 137-701, Republic of Korea
b Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, 505 Banpo-
dong, Seocho-gu, Seoul 137-701, Republic of Korea
ABSTRACT: Synthesis of pyropheophorbide-a-paclitaxel (PPa-PTX) conjugate was performed in high
yield with the aim of searching for an optimal agent for cancer treatment. After synthesis, the conjugate
was confirmed to be linked through an ester bond at the 2′ position of the paclitaxel moiety using multi-
nuclear magnetic resonance spectroscopy. Phototoxicity of PPa and PPa-PTX conjugate, as well as
PTX, was evaluated with three human cancer cell lines (HeLa, CaSki and TC-1). The new conjugate at
0.01–0.06 µM displayed 20–40% higher phototoxicity in HeLa and CaSki cell lines than free PPa and
PTX. Furthermore, cellular uptake of these bio-molecules was examined by confocal laser scanning
microscopy. Although PPa-PTX showed a delayed uptake compared to PPa, it penetrated completely
into cells within 24 h incubation.
KEYWORDS: pyropheophorbide-a, paclitaxel, PDT, photosensitizer, phototoxicity, synthesis.
in cancer chemotherapy [12–14]. It has been approved
INTRODUCTION
for use in the United States against ovarian, breast, and
Photodynamic therapy (PDT) is a promising approach
lung cancers, and Kaposi’s sarcoma [15]. Unfortunately,
for cancer treatment. PDT involves a combination of
when used alone in chemotherapy, it shows serious side-
photosensitizer (PS) with a light in an oxygen rich
effects such as hypersensitivity, nephrotoxicity, and
environment [1, 2]. The advantage of PDT comes out
neurotoxicity [16–20].
preferential uptake of PS by the malignant tissue and
Recent studies reported that porphyrin-platinum and
the ability of PS to destroy a specific region when
porphyrin-cyclodextrin-taxol conjugates exhibit greater
irradiation is supplied [3]. PSs used in PDT are most
antitumor activity and tumor selectivity than parent drugs
often derived from tetrapyrrolic compounds such as
[20–23]. These studies used porphyrin derivatives to
porphyrins, chlorins, phthalocyanines, texaphyrins,
improve the tumor selectivity of the particular anticancer
and bacteriochlorins [4–6]. Of these PSs, chlorin and
drug. The advantage of the combined therapies is their
bacteriochlorin derivatives meet the majority of the
higher therapeutic effect compared to a single therapy
requirements for an ideal PDT PS [7–10]. However, there
approach [23–25].
are challenges to improve their properties for further
Encouraged by these reports, we attempted to
development of PDT [11].
conjugate PTX with a well-studied chlorin derivative,
Paclitaxel (PTX), a complex taxane diterpene, was
pyropheophorbide-a (PPa). In the present study, we
originally isolated from Taxus brevifolia, which is one
of the most effective and widely-used antitumor drugs
describe the synthesis and characterization of the chlorin-
based PTX conjugate prepared through the esterification
reaction using carbodiimide coupling reagent. In addition,
we discuss in vitro phototoxicity and darktoxicity of the
*Correspondence to: Woong Shick Ahn, email: ahnlab1@
catholic.ac.kr, tel: +82 2-2258-6946, fax: +82 2-599-4120
conjugate against three kinds of cancer cells (HeLa,
Copyright © 2012 World Scientific Publishing Company

IN VITRO PHOTOTOXICITY AND DARK-TOXICITY OF A NOVEL SYNTHESIZED PYROPHEOPHORBIDE-A-PACLITAXEL 1007
CaSki, and TC-1) as well as their cellular uptake in TC-1
and HeLa cells.
(DMAP; 3.3 mg, 0.028 mmol) were dissolved in
anhydrous dichloromethane (4 mL). Next, the reaction
mixture was stirred under argon at room temperature in
the dark for 6 h. Progress of the reaction was monitored
by TLC using 2% methanol in dichloromethane until
the starting material was completely consumed. Then,
50 mL of dichloromethane and 100 mL of water were
added to the reaction mixture and the organic layer was
washed with water, 5% hydrochloric acid solution, and
a saturated solution of sodium chloride. The organic
layer was then dried over anhydrous sodium sulfate.
To remove the byproduct dicyclohexylurea (DCU)
formed from DCC, the residue was dissolved in ethyl
acetate and filtered by suction. After that, the solvent
was removed under vacuum and the residue was
purified on silica gel (230–400 mesh) and eluted with
10% acetone in dichloromethane to produce the target
compound. Finally, the product was recrystallized from
dichloromethane-hexane.Yield 29.0 mg (81.5%); brown-
grey solid; Rf: 0.27 (2% methanol in dichloromethane).
Anal. calcd. for C80H83O16N5: C, 70.11; H, 6.10; N, 5.11.
Found: C, 70.30; H, 6.11; N, 5.18. UV-vis (CH2Cl2):
EXPERIMENTAL
General
Column chromatographic separations were performed
over silica gel 60 (63–200 mesh; Merck, Whitehouse
Station, NJ, USA). Analytical thin layer chromatography
(TLC) was carried out on precoated sheets with silica gel
F254 (0.2 mm thick; Merck,). All reactions were carried
out under an argon atmosphere in the dark. The progress
of reactions was monitored by TLC and detection was
carried out using an ultraviolet-lamp at 265 nm or
365 nm. Electronic absorption and fluorescence spectra
were recorded on an Ultrospec®3000 spectrophotometer
(Pharmacia Biotech, Cambridge, England) and
a
RF-5301 spectrofluorophotometer (Shimadzu, Japan).
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Unity Inova 500 (UI 500, Varian,
Palo Alto, CA, USA) spectrometer at 500 MHz; chemical
shifts are expressed in parts per million (ppm) relative
to tetramethylsilane (TMS, 0.00). Elemental analysis and
matrix-assisted laser desorption/ionization (MALDI)
mass spectra were obtained on an EA1112 elemental
analyzer and a Voyager-DETM STR Biospectrometry
Workstation (Applied Biosystems, Carlsbad, CA, USA)
spectrometer. High performance liquid chromatography
(HPLC) spectra were recorded at 660 nm on an Ultimate
3000 (Dionex, Pittsburgh, PA, USA). Confocal laser
microscopy was conducted using a model TCS SP2
apparatus (Leica, Jena, Germany) at 570 nm. All reagents
were purchased from Sigma-Aldrich (St. Louis, MO,
USA), Fluka (St. Louis, MO, USA), Alfa Aesar (Ward
Hill, MA, USA), and Daihan (Seoul, Korea). If necessary,
anhydrous solvents were distilled according to standard
procedures [26]. Other commercially available reagents
were used without further purification. PPa was prepared
from methyl pyropheophorbide-a (MPPa), which
was obtained via methyl pheophorbide-a (MPa) from
Spirulina maxima algae according to previously reported
methods [27, 28]. We prepared PPa using a previously
reported procedure [29]. Yield 1.0 g (88.5%). Rf: 0.25
(5% methanol in dichloromethane). UV-vis (CH2Cl2):
l
max, nm (M-1.cm-1) 667.6 (38291.6), 414.1 (75350.2). 1H
NMR (500 MHz; CDCl3; Me4Si): dH, ppm (ppa stands for
pyropheophorbide-a and tax stands for paclitaxel) 9.48 (s,
1H, 10-meso-Hppa), 9.39 (1H, s, 5-meso-Hppa), 8.52 (1H,
s, 20-meso-Hppa), 8.1 (2H, d, 23-, 27-CHtax, J = 7.02 Hz),
8.01 (1H, dd, 31-CHppa, J = 11.5, 17.8 Hz), 7.64 (2H, d,
39-, 43-CHtax, J = 7.4 Hz), 7.4 (4H, m, 25-, 24-, 26-, and
41-CHtax), 7.3 (2H, d, 33-, 37-CHtax, J = 4.2 Hz), 7.24 (5H,
m, 40-, 42-, 34-, 36-, and 35-CHtax), 6.8 (1H, d, 4′NHtax, J
= 6.7 Hz), 6.28 (1H, s, 10-CHtax), 6.23 (2H, dd, 32-CH2ppa
,
J = 17.9, 11.5 Hz), 6.22 (1H, t, 13-CHtax, J = 8.9 Hz),
5.94 (1H, m, 3′CHtax), 5.67 (1H, d, 2-Ctax, J = 7 Hz), 5.58
(1H, d, 2′CHtax, J = 2.6 Hz), 5.11 (2H, dd, 132-CH2ppa
,
J = 19.5, 19.5 Hz), 4.95 (1H, d, 5-CHtax, J = 7.4 Hz), 4.43
(2H, m, 18-CHppa and 7-CHtax, overlapped), 4.27 (2H, m,
7-CHppa, 20a-CHtax overlapped), 4.18 (1H, d, 20b-CHtax,
J = 8.5 Hz), 3.79 (1H, d, 3-CHtax, J = 7 Hz), 3.69 (2H,
q, 81-CH2ppa, J = 7.5 Hz), 3.62 (3H, s, 121-CH3ppa), 3.40
(3H, s, 21-CH3ppa), 3.24 (3H, s, 71-CH3ppa), 2.61 (2H, m,
172-CH2ppa), 2.56 (1H, s, 7-OHtax), 2.5 (1H, m, 6a-CHtax),
2.41 (3H, s, 29-CH3tax), 2.36 and 2.13 (4H, m, 4-CH2tax
,
171-CH2ppa, overlapped), 2.21 (3H, s, 31-CH3tax), 1.92
(3H, s, 19-CH3tax), 1.88 (1H, s, 1-OHtax), 1.85 (1H, m,
6b-CHtax), 1.75 (3H, d, 181-CH3ppa, J = 7.3 Hz), 1.69 (3H,
t, 82-CH3ppa, J = 7.5 Hz), 1.68 (3H, s, 18-CH3tax), 1.21
(3H, s, 17-CH3tax), 1.12 (3H, s, 16-CH3tax), 0.48 (1H, brs,
21-NHppa), -1.67 (1H, brs, 23-NHppa). 13C NMR (500
MHz; CDCl3): dC, ppm 204.03 (s, C9tax), 196.27 (s, 131-
Cppa), 172.52 (s, 1′-Ctax), 171.41 (s, 173-Cppa), 171.38 (s,
30-Ctax), 170.02 (s, 28-Ctax), 168.18 (s, 21-Ctax), 167.2 (s,
51-Ctax), 159.93 (s, 19-Cppa), 155.61 (s, 16-Cppa), 151.11
(s, 6-Cppa), 149.22 (s, 9-Cppa), 1.37 (s, 14-Cppa), 142.82
(s, 8-Cppa), 141.83 (s, 1-Cppa), 138.12 (s, 11-Cppa), 137.01
(s, 12-Ctax), 136.51 (s, 3-Cppa), 136.42 (s, 4-Cppa), 136.22
(s, 7-Cppa), 133.75 (s, 25-Ctax), 133.71 (s, 32-Ctax), 133.09
l
max, nm (M-1.cm-1) 667.7 (43162.1), 414.2 (80694.4).
1H NMR spectrum of PPa was interpreted as similar
with previously reported in [27, 28]. Proton signals in
1H NMR spectrum for neat Paclitaxel were consistent to
those reported in [30].
Synthesis of pyropheophorbide-a-paclitaxel (PPa-
PTX) conjugate
PPa (14.41 mg, 0.026 mmol), paclitaxel (23 mg,
0.026 mmol), N,N′-dicyclohexylcarbodiimide (DCC;
11.12 mg, 0.053 mmol), and dimethylaminopyridine
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1007–1014

1008 G. BATTOGTOKH ET AL.
(s, 11-Ctax), 132.16 (s, 41-Ctax), 131.82 (s, 2-Cppa), 130.67
(s, 12-Cppa), 130.37 (s, 23, 27-Ctax), 129.46 (s, 22-Ctax),
129.40 (s, 31-Cppa), 129.26 (s, 34, 36-Ctax), 128.90 (s, 24,
26, 40, 42-Ctax), 128.86 (s, 13-Cppa), 128.66 (s, 35-Ctax),
127.22 (s, 33, 37-Ctax), 126.7 (s, 39, 43-Ctax), 122.9 (s,
32-Cppa), 106.14 (s, 15-Cppa), 104.47 (s, 10-Cppa), 97.56
(s, 20-Cppa), 84.69 (s, 5-Ctax), 81.30 (s, 4-Ctax), 79.38
(s, 1-Ctax), 76.66 (s, 20-Ctax), 75.81 (s, 10-Ctax), 75.36
(s, 2-Ctax), 74.37 (s, 2′Ctax), 72.32 (s, 13-Ctax), 72.15 (s,
7-Ctax), 58.74 (s, 8-Ctax), 53 (s, 3′Ctax), 51.62 (s, 17-Cppa),
50.1 (s, 18-Cppa), 48.23 (s, 132-Cppa), 45.83 (s, 3-Ctax),
43.4 (s, 15-Ctax), 35.86 (s, 6-Ctax), 35.77 (s, 14-Ctax), 30.77
(s, 171-Cppa), 29.9 (s, 172-Cppa), 27.04 (s, 17-Ctax), 23.26
(s, 181-Cppa), 22.87 (s, 29-Ctax), 22.37 (s, 16-Ctax), 21.03
(s, 31-Ctax), 19.7 (s, 81-Cppa), 17.6 (s, 82-Cppa), 15.06 (s,
18-Ctax), 12.32 (s, 121-Cppa), 12.25 (s, 21-Cppa), 11.48 (s,
71-Cppa), 9.83 (s, 17-Ctax). MALDI-MS: m/z 1370.5734
(calcd. for [M + H]+ 1370.5835).
points (0.5, 1, 2, 3, 6, 9, 12, 24, 48, and 72 h). The cells
were then washed twice with phosphate buffered saline
(PBS) buffer and stained with 30 µM 4′,6′-diamino-2-
phenylindole (DAPI) solution (500 µL/well) for 10 min at
37 °C. After that, 1 mL of paraformaldehyde (PFA, 1%)
was loaded into each well of 6-well plates for fixation of
cells. After 15 min, the supernatant was discarded and
the coverslip holding cells were put a slide glass where
a drop of mounting solution was placed. After drying for
4 h at room temperature, fluorescence was visualized
using a model TCS SP2 Laser Scanning Spectral Confocal
Microscope (Leica). Fluorescence images were taken at
emission wavelengths of 545 nm and 675 nm. Images of
PPa and PPa-PTX conjugate were assigned as red color.
RESULTS AND DISCUSSION
Synthesis and characterization
Singlet oxygen quantum (SOQ) yields
As shown in Fig. 1, we synthesized the PPa-PTX
conjugate with the intention of searching an optimal
anticancer agent for cancer treatment. Antitumor active
PTX contains three hydroxyl groups at 2′-, 1- and
7-positions, and the hydroxyl group at the 2′ position
displays higher reactivity than the others [5–8, 34].
Therefore, to synthesize the target PPa-PTX conjugate,
we linked the hydroxyl group at the 2′ position of the
PTX moiety and the carboxyl group at the 173 position of
the PPa moiety through an ester bond. Reaction of PPa
with commercial PTX using DCC as a coupling reagent
in the presence of DMAP at room temperature in the
absence of light produced high yields of the target PPa-
PTX conjugate.
SOQ yields were measured by using previously
described techniques in [31, 32]. The SOQ yield (jD)
of each sample was calculated from standard jD value
(PPa - 0.52) [33].
MTT assay
TC-1, positive for HPV E6/E7, HeLa (human
cervix carcinoma cell), and CaSki (a new epidermoid
cervical cancer cell) cell lines were used. For viable
cell counting, the cells (3 × 103 cells) per well (96 well
plate) were treated with PPa, PPa-PTX conjugate, or
PTX at 0.03, 0.06, 0.125, 0.25, 0.5, 1 and 2 µM after
incubation for 24 h (37 °C, 5% CO2) in RPMI 1640
containing 5% fetal bovine serum (FBS). After the cells
were incubated for 24 h, laser irradiation (662 3 nm,
6.25 J/cm2) was performed and the other sample was
kept in dark. Cell growth inhibition was determined a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT)-based assay 24 h after irradiation. For
the MTT assay, 20 µL of 5 mg/mL MTT solution was
added to each cell culture well and cultured for 4 h. Next,
100 µL of dimethylsulfoxide was added to the culture,
shaken for 10 sec, and the absorbance was measured with
a Spectra Max 340 ELISA reader (Molecular Devices,
Sunnyvale, CA, USA) at 570 nm. Measurements were
performed 24 h after laser irradiation. Each group
consisted of three wells; the means of their values were
used as the measured values. The mean SD values were
used for the expression of data.
Subsequently, the structure of PPa-PTX (Fig. 1)
1
1
was fully characterized by H NMR, 2D H-1H COSY,
1H-13C HSQC and 13C NMR techniques, as well as by
MALDI-time of flight-mass spectrometry, UV-vis and
fluorescence spectroscopy, and HPLC.
Figure 2 shows the 1H NMR spectra of neat PTX and
PPa-PTX. After the synthesis of the target compound, the
position of an ester bond bridging PPa and PTX can be
determined as the absence of a resonance corresponding
to either of hydroxyl groups of the PTX moiety for the 1H
NMR spectrum of the final PPa-PTX conjugate. Based
on this, compared to the 1H NMR spectrum of PTX, the
spectrum of PPa-PTX did not show a resonance of the
hydroxyl group at the C2′ that appeared at 3.50 ppm
1
in the H NMR spectrum of neat PTX. The signals of
protons at the C2′ and C3′ positions neighboring the
2′OH group for the PTX moiety shifted downfield (0.79
and 0.15 ppm, respectively) because of a newly formed
ester bond in PPa-PTX. Moreover, a proton (at 2.56 ppm)
at the 7-OH position was determined to be unreacted.
A proton signal of the 1-OH group clearly appeared at
corresponding position (at 1.87 ppm). Multiplets (at 4.43,
2.36 and 2.13 ppm) were observed as overlapping proton
Cellular uptake
Thecells(5×104)inRPMI-1640(2mL)with10%FBS
were seeded into each well of 6-well plate on coverslips
and incubated at 37 °C in a humidified atmosphere of 5%
CO2 for 24 h. Fresh medium containing 2 µM PPa or PPa-
PTX was added, and cells were incubated for various time
Copyright © 2012 World Scientific Publishing Company
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IN VITRO PHOTOTOXICITY AND DARK-TOXICITY OF A NOVEL SYNTHESIZED PYROPHEOPHORBIDE-A-PACLITAXEL 1009
NH
N
NH
N
N
N
(a), (b)
(c)
Spirulina Maxima
Algae
HN
HN
O
O
H3COOC
HOOC
MPPa
PPa
(d)
32
O
29
71
28
31
O
3
5
27
7
6
20
4
O
81
4
O
26
25
8
21
2
82
22
23
21
NH
5
N
O
9
1
20
H
22
21
10
11
OH
7
24
23
HN
24
N
19
HO
14
121
181
12
18
16
14
17
171
13
15
132
131
10
11
O
O
172
O
O
173
C
O
2'
18
34
O
O
30
3'
O
33
31
NH
32
4'
43
42
41
37
38
39
O
35
36
40
PPa-PTX
Fig. 1. Synthesis of pyropheophorbide-a-paclitaxel conjugate. Reagents and conditions: (a) Extraction and acidic treatment; (b)
Methylpheophorbide-a, reflux in collidine for 1.5 h; (c)Acidic treatment with 50% H2SO4 for 2 h at room temperature; (d) Paclitaxel,
DMAP and DCC in dichloromethane at room temperature for 6 h
Fig. 2. Comparison of 1H NMR spectra of PTX and PPa-PTX. (a) 1H NMR spectrum of PTX in CDCl3; (b) 1H NMR spectrum of
PPa-PTX conjugate in CDCl3
Copyright © 2012 World Scientific Publishing Company
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1010 G. BATTOGTOKH ET AL.
The UV absorption and fluorescence spectra of PPa
and PPa-PTX in dimethylsulfoxide (DMSO) were
determined to observe differences by spectrophotometry.
There were no significant differences between the
UV absorption spectra of PPa and PPa-PTX, as shown
in Fig. 3a, but a slightly reduced extinction coefficient
was observed in case of PPa-PTX. This may have been
influenced via the PTX moiety in conjugate. There was
no absorption for the phenyl groups from the PTX moiety
of PPa-PTX.
As illustrated in Fig. 3b, conjugation of PPa and PTX
led to a slight blue shift from 685 nm to 679 nm, and
a noticeable increase in the fluorescence intensity of
PPa-PTX.
Singlet oxygen quantum yield
To determine whether the conjugate is a potential
candidate as a PS in PDT, an indirect method [35] was
utilized to detect a decrease of fluorescence emission
intensity for 9,10-dimethylanthracene (DMA), which
can react with singlet oxygen produced from PS when
irradiation was given. The singlet oxygen yield of the
conjugate was calculated 0.42 which was little bit lower
than that (0.52) of PPa.
Phototoxicity and darktoxicity
We performed an in vitro assay of cell viability on
HeLa, CaSki, and TC-1 cancer cell lines to examine
the phototoxicity and darktoxicity effects of the PPa-
PTX conjugate. The cells (3 × 103 cells/well) were
incubated with PPa, PPa-PTX, or PTX for 24 h, and
photoirradiated with a predetermined light dose (662
3 nm, 6.25 J/cm2). Viability of the cells treated with PPa,
PPa-PTX conjugate, or PTX at various concentrations
(0.01, 0.03, 0.06, 0.12, 0.25, 0.5, 1, and 2 µM) was
determined at 24 h with and without irradiation as
described before [36].
Figure 4 shows the results of phototoxicity and
darktoxicity of PPa, PTX, and PPa-PTX conjugate at
the various concentrations against HeLa, CaSki, and
TC-1 cells. The phototoxicity results of the agents at
0.01–0.25 µM are illustrated.
In case of HeLa cell line (Fig. 4a), PPa-PTX conjugate
at 0.01–0.06 µM produced 50%–20% cell viability,
whereas PPa at the same concentrations produced
around 100%–60% cell viability. These results indicate
that the PPa-PTX conjugate has more than double the
phototoxic effect compared to PPa. However, PPa at a
higher concentration (0.12 µM) was completely toxic,
with no cell survival, which exceeded that of PPa-PTX.
In the darktoxicity test, PPa, PPa-PTX, and PTX showed
negligible effects.
Fig. 3. UV absorption (a) and fluorescence emission (b) spectra
of PPa and PPa-PTX conjugate in DMSO and DMF at same
concentration (1 µM)
signals at C18 and C171 positions of the PPa moiety with
protons at C7 and C14 positions of the PTX moiety. The
proton NMR spectrum demonstrated that an ester bond
formed at 2′ position for the PTX moiety. The other
peaks of PPa and PTX moieties of PPa-PTX in the NMR
spectrum revealed no significant difference and were
easy to produce assignments for. The 13C NMR spectrum
of PPa-PTX was fully cleared up and the carbon atoms
of the PTX moiety were assigned using data previously
reported [30, 35].
The MALDI-TOF mass spectrum of PPa-PTX showed
a peak at m/z = 1370.5734 (100%) for its molecular ion.
The purity of PPa-PTX was determined to be 99.45%
by the analytical reversed-phase HPLC at 660 nm using
methanol/acetone (95:5) as an eluent.
In case of CaSki cells (Fig. 4b), PPa-PTX at 0.01–
0.06 µM showed 80–60% cell viability, while PPa at the
same concentrations produced 100%–95% cell viability.
These results also confirmed that the conjugate had
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1010–1014

IN VITRO PHOTOTOXICITY AND DARK-TOXICITY OF A NOVEL SYNTHESIZED PYROPHEOPHORBIDE-A-PACLITAXEL 1011
Fig. 4. Comparison of results of PDT and darktoxicity of PPa, PTX and PPa-PTX conjugate at various concentration range
(0.03–2 µM) in three cell lines; HeLa (a), CaSki (b) and TC-1 (c). Cells were incubated with PSs or PTX in 10% serum containing
medium for 24 h and then exposed to light (662.5 nm) for a total fluence of 6.25 J/cm2. In case of darktoxicity, irradiation was not
given. After treatment, the cells were incubated in growth medium for 24 h. The data are expressed as mean of three experiments.
Error bars present standard deviation
20%–40% higher phototoxicity than free PPa at same
condition. PPa at 0.25 µM produced no cell viability. In
the darktoxicity test, PPa, PPa-PTX, and PTX showed
negligible effects.
IncaseoftheTC-1cellline(Fig.4c),PPa-PTXconjugate
at 0.01–0.06 µM produced 80%–40% cell viability, while
PPa at the same concentrations produced 100%–0% cell
viability. The data indicated that the conjugate had a lower
phototoxicity, except 0.01 µM, than free PPa in this cell
line. In the darktoxicity test, PPa-PTX showed similar
cytototoxicity with PTX at all concentrations.
The three cell lines were affected differently by free
PPa and PPa-PTX when irradiated. HeLa and CaSki cells
were 30%–40% more sensitive for PPa-PTX at 0.01–
0.06 µM than free PPa, whereas, TC-1 cells were more
sensitive to free PPa than the others. As reported [37–39],
free PPa is 100% phototoxic at concentrations exceeding
0.15 µM in several cancer cell lines, when the same
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1011–1014

1012 G. BATTOGTOKH ET AL.
laser power was used. In addition, some pheophorbide-a
derivatives are 50% phototoxic in different cell lines at
0.07–1.5 µM [40–42].
The results indicate some improvements using the PPa-
PTX conjugate, especially in HeLa and CaSki cell lines.
This enhancement might be related to a combination of
PS and anticancer drug.
Cellular uptake
As shown in Fig. 5, the cellular uptake of PPa and PPa-
PTX (2 µM) in the presence of DAPI in TC-1 (Fig. 5a)
and HeLa (Fig. 5b) cells at different time points (0.5, 1,
2, 3, 6, 9, 12, 24, 48, and 72 h) was examined to observe
time-dependent penetration of the conjugate into the cells
Fig. 5. Comparison of cellular uptake results (1000 ×) for 2 µM of free PPa and PPa-PTX conjugate in TC-1 cells (a) and HeLa
cells (b) after different points of time (3, 6, 12, 24, and 48 h) by confocal microscopy. DAPI, which gives blue fluorescence, was
used as a staining probe for cell nucleus. Differential interference contrast (DIC) denotes a picture was taken without fluorescence.
Red fluorescence refers to presence of PS
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J. Porphyrins Phthalocyanines 2012; 16: 1012–1014

IN VITRO PHOTOTOXICITY AND DARK-TOXICITY OF A NOVEL SYNTHESIZED PYROPHEOPHORBIDE-A-PACLITAXEL 1013
by confocal microscopy. Here, we have chosen images of
cells treated with PPa and PPa-PTX taken after 3, 6, 12,
24, and 48 h of incubation.
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The results confirmed that the uptake of PPa-PTX into
TC-1 cell began after 6 h incubation, whereas the uptake
of PPa started later, at 3 h incubation (Fig. 5a). Moreover,
in case of HeLa cell, uptake of PPa-PTX conjugate began
after 9 h incubation, while uptake of PPa started after 6 h
of incubation (Fig. 5b). The uptake of PPa and PPa-PTX
in HeLa cells was slightly slower than that in TC-1 cells.
However, maximum uptake occurred in both cells 24 h after
adding PS. The collective observations support the view that
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24 h. The late uptake of the PPa-PTX conjugate could be
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In summary, we have synthesized and characterized
a novel PPa-PTX conjugate. Its photoxicity and
darktoxicity were evaluated on three kinds of cancer cell
lines (HeLa, CaSki, and TC-1) by an established MTT
assay. PPa-PTX at lower concentrations (0.01–0.06 µM)
displayed better phototoxicity in HeLa and CaSki cell
lines compared to PPa. Cellular uptake of the conjugate
and PPa was also examined in HeLa and TC-1 cells;
PPa-PTX conjugate fully entered the cells within 24 h.
Further studies will focus on the cellular localization of
PPa and PPa-PTX to test whether the efficacy difference
relates to PS localization in the cells, and whether the
higher phototoxicity of the conjugate at lower dosage
in HeLa and TC-1 cells is due to the over-expression of
the ABCG2 protein. As well, in vivo experiments will be
conducted to address the hypothesis that the conjugate
may show more phototoxicity even at higher dosage than
PPa because there is a fact that in vitro efficacy of PPa
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Acknowledgements
The study was supported by The Catholic Harvard
Wellman Photomedicine Core Technique Development
Center funded by the Ministry of Education, Science
and Technology, Seoul, Republic of Korea (Grant
No. 5-2012-A0154-00001). The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
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