Preparation and sonodynamic activities of water-soluble tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine and its bovine serum albumin conjugate

Article abstract of DOI:10.1016/j.ultsonch.2014.05.019

Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine, namely tetra-α-(3- carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC₄), has been prepared and characterized. The interactions between ZnPcC₄ and bovine serum albumin (BSA) were also investigated by absorption and fluorescence spectroscopy. It was found that there were strong interactions between ZnPcC₄ and BSA with a binding constant of 6.83 × 10⁷ M^-1. A non-covalent BSA conjugate of ZnPcC₄ (ZnPcC ₄-BSA) was prepared. Both ZnPcC₄ and ZnPcC₄-BSA exhibited efficient sonodynamic activities against HepG2 human hepatocarcinoma cells. Compared with ZnPcC₄, conjugate ZnPcC₄-BSA showed a higher sonodynamic activity with an IC₅₀ value of 7.5 μM. Upon illumination with ultrasound, ZnPcC₄-BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized with carboxyls can serve as promising sonosensitizers for sonodynamic therapy.

Full text of DOI:10.1016/j.ultsonch.2014.05.019

Ultrasonics Sonochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson
Preparation and sonodynamic activities of water-soluble
tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalocyanine
and its bovine serum albumin conjugate
1
1
⇑
He-Nan Xu , Hai-Jun Chen , Bi-Yuan Zheng, Yun-Quan Zheng, Mei-Rong Ke, Jian-Dong Huang
College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of
ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine,
namely tetra-a-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4), has been prepared and character-
ized. The interactions between ZnPcC4 and bovine serum albumin (BSA) were also investigated by
absorption and fluorescence spectroscopy. It was found that there were strong interactions between
ZnPcC4 and BSA with a binding constant of 6.83 ꢀ 107 Mꢁ1. A non-covalent BSA conjugate of ZnPcC4
(ZnPcC4–BSA) was prepared. Both ZnPcC4 and ZnPcC4–BSA exhibited efficient sonodynamic activities
against HepG2 human hepatocarcinoma cells. Compared with ZnPcC4, conjugate ZnPcC4–BSA showed a
Received 1 April 2014
Received in revised form 23 May 2014
Accepted 23 May 2014
Available online xxxx
Keywords:
Phthalocyanine
Serum albumin
Ultrasound
higher sonodynamic activity with an IC50 value of 7.5 lM. Upon illumination with ultrasound, ZnPcC4–
BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular
apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized
with carboxyls can serve as promising sonosensitizers for sonodynamic therapy.
Ó 2014 Elsevier B.V. All rights reserved.
Sonodynamic therapy
Sonosensitizer
1. Introduction
sonosensitizers are porphyrin derivatives such as hematoporphy-
rin (the first generation photosensitizer), portoporphyrin IX, and
Sonodynamic therapy (SDT) is a new approach for cancer treat-
ment, which was developed on the basis of photodynamic therapy
(PDT) [1–3]. PDT utilizes the combined action of photosensitizer,
light, and molecular oxygen to eradicate cancer cells [4,5]. How-
ever, clinical application of PDT is limited to the treatment of
superficial and small solid tumors due to the poor tissue penetra-
tion of excitation light [6]. Similarly to PDT, SDT utilizes the com-
bined action of a sonosensitizer and low-intensity ultrasound to
cause cell and tissue damage. Ultrasound has an appropriate tissue
attenuation that lets it penetrate and reach deep-seated tissues
without losing ability to focus energy into small volume. This
unique advantage makes SDT more efficient for noninvasive treat-
ment of deep-seated tumors compared with PDT [7–10]. Accumu-
lating evidences indicate that SDT has a great potential in cancer
therapy [11–15].
ATX-70 (a gallium porphyrin complex) [7,16]. Nonetheless, these
porphyrin-based sonosensitizers have some shortcomings such as
skin photosensitivity and unsatisfactory sonocytotoxicity. Signifi-
cant efforts have therefore been put in new sonosensitizers which
have better sonochemical properties, higher tumor specificity, and
less skin photosensitivity.
Phthalocyanines have been proposed as highly promising
photosensitizers over the last three decades, owing to their intense
absorption in the red visible region, high efficiency to generate
reactive oxygen species, low dark toxicity, and low skin photosen-
sitivity [17–19]. However, the use of phthalocyanines as sonosen-
sitizers for SDT remains relatively little studied [20–23]. Herein, we
reported the preparation and in vitro sonodynamic activities of a
tetra-a-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4)
and its bovine serum albumin conjugate (ZnPcC4–BSA). Zinc(II)
phthalocyanines functionalized with carboxyl(s) have been used
as efficient photosensitizers, due to their water solubility and high
singlet oxygen yield [24–26]. However, the sonodynamic activity
of this type of phthalocyanine has not been investigated. In addi-
tion, despite recent advances using serum albumin as the protein
carrier for anticancer drugs or photosensitizers to improve their
The overall efficacy of sonodynamic therapy depends greatly on
the behavior of the sonosensitizer. To date, the most common
⇑
Corresponding author. Tel.: +86 591 22866235; fax: +86 591 22866227.
E-mail address: jdhuang@fzu.edu.cn (J.-D. Huang).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ultsonch.2014.05.019
1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

2
H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
passive targeting properties [27–29], to the best of our knowledge,
the application of serum albumin for delivery of sonosensitizer has
no precedents in the literature.
then removed in vacuum and the residue was treated with water
and acidified with HCl aqueous solution (1 M) to induce precipita-
tion. The precipitate was collected by filtration, washed with water
until pH 7, and dried in vacuum. The crude product was subjected
to column chromatography using DMF/ethyl acetate (1:3, v/v) and
then DMF as eluents. The last green fraction was collected, and
dried in vacuum to give dark green solid (0.10 g, 37%). Rf = 0.67
(MeOH). IR (KBr, cmꢁ1): 3273.8 (O–H); 1702.1 (C@O); 3065.5
(Ar–H), 1577.3, 1481.1, 1440.2 (C@C, Ar); 1247.2 (Ar–O–Ar);
1129.7 (C–N). MS (ESI): m/z 1121.2 [M+H]+. 1H NMR (DMSO-d6,
ppm): d 9.10–9.28 (m, 2H), 8.68–8.75 (m, 1H), 8.55 (t, J = 8.2 Hz,
1H), 8.20 (t, J = 7.6 Hz, 1H), 8.07 (t, J = 7.6 Hz, 2H), 7.84–7.92 (m,
4H), 7.63–7.73 (m, 17H). Anal. Calcd for C60H32N8O12Zn: C, 64.21;
H, 2.87; N, 9.98. Found: C, 64.03; H, 3.01; N, 9.82.
2. Experimental
2.1. General
All the reactions were performed under an atmosphere of nitro-
gen. N,N-dimethylformamide (DMF) was dried over molecular
sieves and further distilled under reduced pressure before use.
Potassium carbonate was activated by muffle at 300 °C under nor-
mal pressure. Chromatographic purifications were performed on
silica gel columns (100–200 mesh, Qingdao Haiyang Chemical
Co., Ltd., China) with the indicated eluents. Bovine serum albumin
was purchased from Sigma–Aldrich Co. All other solvents and
reagents were of reagent grade and used as received.
2.3. Preparation of phthalocyanine-BSA conjugate
The non-covalent BSA conjugate of ZnPcC4 was prepared
according to the literature procedure [33]. The conjugate was
obtained by stirring a mixture of ZnPcC4 and BSA (molar ratio:
ZnPcC4/BSA = 2/1) in a phosphate buffered saline (PBS) at ambient
temperature overnight, followed by gel chromatography on a
G-100 Sephadex column using deionized water as eluent. The con-
jugate ZnPcC4–BSA collected as the first blue fraction was lyophi-
lized to remove water. The protein content in the conjugate was
calculated from the absorbance at 280 nm in a diluted PBS solution
(pH = 7.4) with reference to the corresponding molar absorptivity
1H NMR spectra were recorded on a Bruker AVANCEIII 400 spec-
trometer (400 MHz). Chemical shifts were relative to internal SiMe4
(d = 0 ppm). Mass spectra were recorded on a Finnigan LCQ Deca
xpMAX mass spectrometer. IR spectra were recorded on a Perkin-
Elmer SP2000 FT-IR spectrometer, using KBr disks. Elemental anal-
yses were performed by Element Vario EL III equipment. Electronic
absorption spectra were measured on a Shimadzu UV-2450 UV–
vis spectrophotometer. Fluorescence spectra were taken on an Edin-
burgh FL900/FS900 spectrofluorometer. The fluorescence quantum
yields (UF) were determined by the equation: UF(sample) = (ns2ample
/
of BSA (
e
= 4.85 ꢀ 104 Mꢁ1 cmꢁ1). The phthalocyanine concentra-
n2ref)ꢂ(Fsample/Fref)ꢂ(Aref/Asample
) UF(ref), where F, A, and n are the mea-
tion was calculated from the Q band absorbance in a diluted
sured fluorescence (area under the emission peak), the absorbance
at the excitation position, and the refractive index of the solvent,
respectively. Unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF
DMF solution with reference to the corresponding molar absorptiv-
ity (
e
= 2.19 ꢀ 105 Mꢁ1 cmꢁ1).
[
UF(ref) = 0.28] was used as the reference [30]. The singlet oxygen
2.4. In vitro studies
quantum yields (UD) was measured in DMF by a steady-state
method using 1,3-diphenylisobenzofuran (DPBF) as the scavenger
and ZnPc [UD(ref) = 0.56] as reference [31,32].
For in vitro studies, ZnPcC4 was first dissolved in DMF (1.0 mM)
and the solution was diluted to 80 lM with 0.5% (wt.) aqueous
solution of Cremophor EL (Sigma, 0.5 g in 100 mL of water).
ZnPcC4–BSA conjugate was dissolved in PBS with a concentration
2.2. Synthesis of ZnPcC4
of 80
lM. BSA was also dissolved in PBS with a concentration of
80 M. These solutions were clarified with 0.45
diluted with the cellular culture medium (as described below) to
l
l
m filter, and then
2.2.1. 3-(3-carboxyphenoxyl)phthalonitrile (a-C)
A mixture of 3-nitrophthalonitrile (0.87 g, 5 mmol), 3-hydroxy-
benzoic acid (0.69 g, 5 mmol), and anhydrous K2CO3 (2.07 g,
15 mmol) in dry DMSO (20 mL) was stirred at room temperature
for 24 h under nitrogen atmosphere. The reaction mixture was fil-
tered by sand core funnel and the filtrate was poured into ice water
(200 mL). Subsequently, HCl aqueous solution (2 M) was added to
the filtrate until pH = 1–3 to give white precipitate, which was col-
lected by filtration, washed with water until pH = 7 and dried in
vacuum. The crude product was purified by recrystallization with
DMF/water to afford white solid (1.11 g, 84%). Rf = 0.53 (EtOH). IR
(KBr, cmꢁ1): 3078.4 (Ar–H); 1585.6, 1466.7, 1450.9 (C@C, Ar);
1303.7, 1278.7, 1208.9 (Ar–O–Ar); 2232.9 (C„N); 1688.8 (C@O).
MS (ESI): m/z 263.1 [MꢁH]ꢁ. 1H NMR (DMSO-d6, ppm): d 13.16
(br., 1H), 7.81–7.88 (m, 3H), 7.62–7.67 (m, 2H), 7.52 (t, J = 0.6 Hz,
1H), 7.32 (d, J = 4.2 Hz, 1H). Anal. Calcd for C15H8N2O3: C, 68.18;
H, 3.05; N, 10.60. Found: C, 67.99; H, 3.27; N, 10.86.
appropriate concentrations.
Human hepatocellular carcinoma HepG2 cells were obtained
from the cell bank of the Chinese Academy of Science, Shanghai,
China. The cells were maintained in RPMI 1640 medium (Invitro-
gen) supplemented with 10% fetal calf serum, streptomycin
(50 l
g mLꢁ1), and penicillin (50 units mLꢁ1). The cells were incu-
bated at 37 °C in a humidified CO2 (5%) incubator, and the medium
was refreshed every 1–2 days. Cells in the exponential phase of
growth were used in the following experiments.
2.4.1. Sonodynamic activity assay
HepG2 cells were exposed to ultrasound after an incubation of
45 min with ZnPcC4–BSA, ZnPcC4, and BSA, respectively. The exper-
imental set-up for ultrasound exposure is showed in Fig. 1. The
transducer with a diameter of 45 mm was submerged in the stain-
less steel container filled with cold degassed water. Polystyrene
tube containing 0.5 mL of cell suspension (2 ꢀ 105 cells mLꢁ1 in
RPMI 1640 medium) was fixed vertically on the focal area of the
transducer. The distance between the bottom of the polystyrene
tube and the transducer was 1 cm. The spatial average ultrasonic
intensity was 2.0 W cmꢁ2 with a frequency of 1.0 MHz in continu-
ous waves and the ultrasonic time was set at 3 min. The ultrasound
system (Therapy Ultrasound 4150) was manufactured by the
CARCI Company. For all experiments, the cold degassed water
was used as the ultrasonic coupling medium, thereby reducing
2.2.2. 1,8(11),15(18),22(25)-tetrakis-(3-carboxyphenoxyl) zinc(II)
phthalocyanine (ZnPcC4)
A
mixture of 3-(3-carboxyphenoxyl)phthalonitrile (0.26 g,
1.0 mmol) and K2CO3 (0.14 g, 1.0 mmol) in n-pentanol (20 mL)
was stirred at 90 °C under nitrogen atmosphere for 15 min, and
then zinc acetate (0.11 g, 0.6 mmol) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) (0.4 mL, 2.6 mmol) were added. The
mixture was heated to reflux at 130 °C for 10 h. The solvent was
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
3
cell-permeant compound, is deacetylated by intracellular esterase
and converted to the reduced probe dichlorodihydrofluorescein
(DCFH), which can be rapidly oxidized to the highly fluorescent
product DCF in the presence of ROS. After SDT treatment, HepG2
cells were incubated with DCFH-DA (10 lM, dilute in serum-free
medium) for 2 h at 37 °C under 5% CO2. The cells were then washed
with PBS and analyzed immediately by flow cytometer through
FL-1 filter with an excitation wavelength of 488 nm.
2.4.4. Cellular uptake
HepG2 cells (2 ꢀ 105) were incubated with a solution of ZnPcC4
or ZnPcC4–BSA in the medium (10 lM, 1 mL) for 0, 15, 30, 45, 60,
and 75 min, respectively. In each time point, the cells were col-
lected by centrifugation (3 min, 3000 rpm) and washed with PBS.
After removing the PBS, the cells were lyzed with DMF (2 mL).
The mixture was sonicated for 10 min and then centrifuged at
15,000 rpm for 5 min. The fluorescence spectrum of the superna-
tant was recorded in an Edinburgh FL900/FS900 spectrofluorome-
ter (excited at 610 nm and monitored emission at 630–800 nm).
From the area of emission peak, the concentration of ZnPcC4 in
the solution was determined by comparison with a calibration
curve obtained with standard solutions of ZnPcC4. Each experiment
was repeated three times.
Fig. 1. Diagram of ultrasonic exposure apparatus.
thermal effect caused by ultrasound irradiation. All experiments
were randomly divided into eight groups: ZnPcC4–BSA mediated
SDT, ZnPcC4 mediated SDT, BSA mediated SDT, ultrasound treat-
ment alone, and the controls including ZnPcC4–BSA, ZnPcC4, and
BSA treatment alone and sham group. For the sham group, the cells
were not treated neither by ultrasound nor any compound incuba-
tion. All the experiments were performed in the dark.
2.5. Statistics
The 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT) assay was used to determine the cell viability. After
SDT treatment, the treated cells or control cells (100 lL) were
Data were expressed as mean standard deviation (SD). Statis-
tical analyses were performed using Student’s t-test. P values less
than 0.05 were considered statistically significant.
seeded on a 96-well plate and incubated overnight at 37 °C under
5% CO2. The medium was then removed from each well. After that,
a 10 l lL of the med-
L of MTT solution (5 mg mLꢁ1 in PBS) and 90
ium were added into each well, followed by incubation for 4 h at
37 °C under 5% CO2. Subsequently, the MTT-containing medium
3. Results and discussion
was removed and 150 lL DMSO was added into each well. After
3.1. Synthesis and photophysical properties of ZnPcC4
shaking for 10 min, the optical density (OD) at 490 nm was mea-
sured using microplate reader (MULTISKAN MK3, Thermo SCIEN-
TIFIC). The killing rate was calculated using the following equation:
Scheme 1 shows the synthetic route of tetra-
oxyl) zinc(II) phthalocyanine (ZnPcC4). Firstly,
a
-(3-carboxyphen-
a
nucleophilic
substitution reaction occurred between 3-nitrophthalonitrile and
3-hydroxybenzoic acid under alkaline condition to give the precursor
3-(3-carboxylphenoxy)phthalonitrile (a-C). The precursor a-C was
Cytotoxicity ð%Þ ¼ ðOD sham group ꢁ OD treatment groupÞ
=OD sham group ꢀ 100%:
further underwent a cyclotetramerization in the presence of zinc
acetate and K2CO3 in n-pentanol using DBU as a catalyst to afford
the tetra-substituted phthalocyanine ZnPcC4. As reported previ-
ously [25,35], anhydrous K2CO3 was added to inhibit the possible
esterification between the carboxylic groups and the solvent of
n-pentanol. The prepared compounds were characterized by 1H
NMR, MS, elemental analyses, and FT-IR spectroscopic methods.
The photophysical and photochemical properties of ZnPcC4
were measured in DMF. As shown in Fig. 2A, ZnPcC4 gave a typical
absorption spectrum of non-aggregated phthalocyanines, exhibit-
ing a sharp and intense Q-band at 688 nm. Upon excitation at
610 nm, it showed a strong fluorescence at 702 nm with a fluores-
cence quantum yield (UF) of 0.11 relative to unsubstituted zinc(II)
phthalocyanine (ZnPc) (UF = 0.28) (Fig. 2B and Table 1). To evaluate
the photosensitizing efficiency of ZnPcC4, its singlet oxygen quan-
tum yields (UD) was also determined by a steady-state method
with 1,3-diphenylisobenzofuran (DPBF) as the scavenger. It was
found that ZnPcC4 is an excellent singlet oxygen generator with a
UD value of 0.53. The spectroscopic behavior of ZnPcC4 was further
investigated in aqueous solutions. As shown in Fig. 2A, two weak
and broad peaks at 651 nm and 697 nm were observed for ZnPcC4
in PBS solution, meanwhile, its fluorescence intensity was very
weak relative to that in DMF (Fig. 2B), indicating that ZnPcC4 is
aggregated in PBS [26].
2.4.2. Apoptosis
After SDT treatment, HepG2 cells were seeded in 6-multiwell
plates and incubated for 24 h at 37 °C under 5% CO2. Apoptosis
was then analyzed using a flow cytometer (Coulter EpicsXL Beck-
man, USA) with Annexin V-FITC (fluorescein isothiocyanate) and
propidium iodide (PI) staining. Apoptosis Detection Kit (KeyGEN
BioTECH, Nanjing, China) was used in the present study. The
treated cells were trypsinized to obtain single cell suspension
and washed twice with PBS. The obtained cell pellets were re-
suspended in 500
a mixture of Annexin V-FITC (5
and then incubated at 37 °C for 15 min. After that, the samples
were analyzed using the flow cytometer.
l
L of the binding buffer, and then added with
L) and PI (5 L) working solution
l
l
2.4.3. Evaluation of reactive oxygen species
Intracellular reactive oxygen species (ROS) production was
assessed with a flow cytometer by measuring the fluorescence
intensity of dichlorofluorescein (DCF) as described by other
researchers [34]. The probe 2,7-dichlorodihydrofluorescein diace-
tate (DCFH-DA) was purchased from Sigma and dissolved in DMSO
to produce a stock solution (1 mM). DCFH-DA, a non-fluorescent
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

4
H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
COOH
COOH
O
N
COOH
CN
CN
O
N
HO
N
Zn
N
CN
CN
K2CO3, Zn(OAc)2
N
N
DBU, n-pentanol,
reflux
K2CO3, DMSO, r.t.
O
COOH
N
N
O
NO2
O
-C
a
COOH
HOOC
ZnPcC4
Scheme 1. Synthesis of ZnPcC4.
Table 1
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A
Photophysical data of ZnPcC4 and ZnPcC4–BSA.
ZnPcC
ZnPcC
ZnPcC
4 in DMF
ꢀ 105
UF
UD
a
b
c
Compounds
Solvents
kabs
(nm)
kem
e
(nm)
(Mꢁ1ꢂcmꢁ1
)
4 in PBS
ZnPcC4
DMF
PBS
688
651, 697
702
711
2.19
–
0.11
0.01
0.53
–
4-BSA in PBS
ZnPcC4–BSA
PBS
651, 701
711
–
0.02
–
a
b
c
Excited at 610 nm.
Using ZnPc in DMF as the reference (UF = 0.28) [30].
Determined by using DPBF as chemical quencher, and using ZnPc in DMF as the
reference (UD = 0.56) [31,32].
presence of the quencher respectively, CQ is the concentration of
the quencher, KA is the binding constant, and n is the binding site
number. The binding constant KA and binding site number were
found to be 6.83 ꢀ 107 Mꢁ1 and 1, respectively:
300
400
500
600
700
800
Wavelength (nm)
B
15000
10000
5000
0
ZnPcC4
in D
MF
F0 ꢁ F
lg
¼ lg KA þ n lg CQ
ð1Þ
ZnPcC
4 in PBS
4-BSA in PBS
F
ZnPcC
On the other hand, upon addition of BSA, the absorption peak at
652 nm of ZnPcC4 in PBS reduced in intensity gradually, while the
Q band at 696 nm correspondingly increased in intensity (Fig. 3B).
Both this observation and the high KA indicated that there was a
strong interaction between ZnPcC4 and BSA. Furthermore, the Job’s
plot with titrations (kabs = 699 nm) at the different concentrations
of BSA exhibited a maximum at about 0.5 mol fraction (see the
inset in Fig. 3B). This indicates that ZnPcC4 conjugated to BSA with
a 1:1 stoichiometry.
660 680 700 720 740 760 780 800
In view of the strong interaction between ZnPcC4 and BSA,
attempts were made to prepare their non-covalent conjugate
according to the literature procedure [33]. The conjugate was
obtained by stirring a mixture of ZnPcC4 and BSA (with a molar
ratio of 2) in PBS at ambient temperature overnight, followed by
a gel chromatography separation step. The molar ratio of phthalo-
cyanine to BSA was found to be 1:1 for the obtained ZnPcC4–BSA
conjugate, which is in accord with the result from the Job’s plot.
The non-covalent conjugate ZnPcC4–BSA was readily soluble in
water. Therefore, its spectroscopic properties were measured in
PBS. As shown in Fig. 2A, the Q-band of ZnPcC4–BSA conjugate
showed an obvious increase in intensity at 699 nm with slight
red-shift compared to that of ZnPcC4. Accordingly, the fluorescence
intensity of ZnPcC4–BSA conjugate at 711 nm in PBS was higher
than that of ZnPcC4 (Fig. 2B). Moreover, it is need to mention that
the spectrum of ZnPcC4–BSA remained virtually unchanged upon
standing for long time. These findings strongly suggest that ZnPcC4
is relatively non-aggregated in the protein cavity and ZnPcC4–BSA
conjugate is stable in PBS solution.
Wavelength (nm)
Fig. 2. (A) Electronic absorption and (B) fluorescence emission spectra (excited at
610 nm) of ZnPcC4 and ZnPcC4–BSA (both at 3 M) in DMF and PBS.
l
3.2. Interaction and conjugate of ZnPcC4 with BSA
BSA has been used as a carrier for targeted delivery of various
anti-cancer drugs [29]. To enhance the biocompatibility and selec-
tivity of phthalocyanine-based sensitizers, attempts were made to
prepare the BSA conjugate of ZnPcC4. We firstly analyzed the inter-
actions of ZnPcC4 with BSA by a fluorescence quenching method
[36–38]. Fig. 3A shows the change in the fluorescence spectra of
BSA in PBS upon titration with ZnPcC4. With the increasing of
ZnPcC4, the intrinsic emission band of BSA at 340 nm decreases
in intensity. As shown in the inset of Fig. 3A, the quenching data
follow the double logarithmic equation (Eq. (1)) [36,39], where
F0 and F are the intensities of fluorescence in the absence and
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
5
1.0
0.5
A
6000
4000
2000
0
0.0
-0.5
-6.3 -6.0 -5.7 -5.4 -5.1
log[c]
320
360
400
440
Wavelength (nm)
0.16
0.12
0.08
0.04
0.00
0.4
0.3
0.2
0.1
0.0
B
699 nm [ BSA ]
30 mM
0.0
0.2
0.4
0.6
0.8
1.0
[ZnPcC ] / ([ZnPcC ]+[BSA])
0 mM
4
4
652 nm
696 nm
500
600
700
800
Wavelength (nm)
Fig. 3. (A) Change in fluorescence spectrum of BSA (2
lM, excited at 280 nm) in PBS
upon titration with ZnPcC4. The inset shows the corresponding lg[(F0 ꢁ F)/F] vs.
lg[ZnPcC4] plot. (B) Changes in the absorption spectrum of ZnPcC4 (2 M) in PBS
l
upon titration with BSA. The inset shows the Job’s plot for the binding of ZnPcC4
and BSA (kabs = 699 nm).
Fig. 4. Effects of ZnPcC4, ZnPcC4–BSA, and BSA on HepG2 cells in the absence and
presence of ultrasound (1 MHz, 2.0 W cmꢁ2
,
3 min). Data are expressed as
mean SD (n P 6). (A) The cells were incubated with ZnPcC4, ZnPcC4–BSA, and
BSA at the concentration of 10 M for 45 min before ultrasound illumination. (B)
The cells were incubated with ZnPcC4–BSA at the concentration of 0, 5, 10, and
15 M for 45 min before ultrasound illumination (p < 0.05 vs. untreated control).
l
3.3. In vitro sonodynamic anticancer activities
l
The effects of ZnPcC4, BSA, and ZnPcC4–BSA against HepG2 cells
were examined in the absence and presence of ultrasound. In the
absence of ultrasound, ZnPcC4, ZnPcC4–BSA, and BSA (all at
10 lM) did not exhibited apparent cytotoxicity on HepG2 cells,
as shown in Fig. 4A. When ultrasound treatment alone was used,
the cell viability was found to be 86.6%. This suggests ultrasound
treatment alone caused slight cellular damage. In the presence of
ultrasound, the cell viability caused by BSA was found to be
82.3%, which is comparable with that induced by ultrasound treat-
ment alone (82.3% vs. 86.6%, p > 0.05). This indicates that BSA
hardly presents sonodynamic activity against HepG2 cells. When
HepG2 cells were treated with ZnPcC4 (10
lM) and ZnPcC4–BSA
(10 M) in the presence of ultrasound, the cell viability decreased
l
to 52.0% and 22.9%, respectively. Clearly, both ZnPcC4 and ZnPcC4–
BSA exhibited significant sonocytotoxicities toward HepG2 cells
(p < 0.05 vs. ZnPcC4 alone, ZnPcC4–BSA alone, or ultrasound treat-
ment alone). By comparison, ZnPcC4–BSA showed a higher sonody-
namic activity than ZnPcC4 (p < 0.05). Further, the concentration-
dependent sonocytotoxicity of ZnPcC4–BSA toward HepG2 cells
was determined after 45 min of incubation and subsequent illumi-
nation with ultrasound. From the dose response curve (Fig. 4B), the
IC50 value, defined as the sonosensitizer concentration required to
Fig. 5. Cellular uptakes of ZnPcC4 (10 lM) and ZnPcC4–BSA (10 lM) by HepG2 cells
after 45 min of incubation.
To account for the different sonodynamic activities of ZnPcC4
and ZnPcC4–BSA, their cellular uptakes were compared by an
extraction method. In this method, DMF was used to lyze the cells
and extract the photosensitizer. Since the phthalocyanine can be
released from BSA cage in DMF, the relative fluorescence intensity
of extraction solution can reflect the difference of cellular uptake
kill 50% of cells, was calculated to be 7.5 lM for ZnPcC4–BSA.
These results indicate that the photosensitizer ZnPcC4 and its
BSA conjugate can serve as efficient sonosensitizers for SDT.
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

6
H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
Fig. 6. Flow cytometric analysis of the cell death mechanism induced by SDT treatment (1.0 MHz, 2.0 W cmꢁ2, 3 min) on HepG2 cells with Annexin V-FITC and PI staining.
Fluorescence intensity for Annexin V-FITC is plotted on the X-axis, and fluorescence intensity for PI is plotted on the Y-axis. The bottom right quadrant of each panel shows the
early apoptotic cells (Annexin V-FITC positive, PI negative), while the top right quadrant of each panel shows the late apoptotic or necrotic cells (Annexin V-FITC positive, PI
positive). (A) Control group (ZnPcC4–BSA alone) and (B) ZnPcC4–BSA-mediated SDT.
Fig. 7. Flow cytometric analysis of intracellular ROS induced by SDT treatment (1.0 MHz, 2.0 W cmꢁ2, 3 min) on HepG2 cells with DCFH-DA staining. Fluorescence intensity of
DCF is plotted on the X-axis, and the number of the cells is plotted on the Y-axis. (A) Control group (ultrasound alone) and (B) ZnPcC4–BSA-mediated SDT.
between ZnPcC4 and ZnPcC4–BSA. Cellular uptake of ZnPcC4 and
ZnPcC4–BSA by HepG2 cells were rapid, which occurred after incu-
bation for 15 min and reached a plateau at 45 min. No further sig-
nificant uptake was observed over the subsequent 75 min. As
shown Fig. 5, after 45 min of incubation, ZnPcC4–BSA conjugate
showed about 1.6-fold higher cellular uptake toward HepG2 cells
than the free ZnPcC4, which probably results from the high affinity
of cancer cells for albumin [28]. Therefore, the higher sonocytotox-
icity of ZnPcC4–BSA compared with ZnPcC4 can be mainly attrib-
uted to its higher cellular uptake.
As shown in Fig. 7, the fluorescence curves for cells treated by
ZnPcC4–BSA-mediated SDT shift to the right compared with that
for cells of control group. This indicates that ZnPcC4–BSA caused
an obvious increase of ROS generation in the presence of ultra-
sound. Therefore, it can be reasoned that ZnPcC4–BSA, upon
excited by ultrasound, can induce the generation of intracellular
ROS, which next leads to the apoptosis or/and necrosis of cancer
cells. However, the sonochemical mechanisms of ROS formation
induced by ZnPcC4–BSA need further investigation.
4. Conclusions
3.4. Apoptosis and intracellular reactive oxygen species (ROS)
A new zinc(II) phthalocyanine tetra-substituted with carboxyl
groups at
a-positions and its BSA conjugate were prepared and
In order to determinate the possible mechanism of sonodynam-
characterized. The compound ZnPcC4 is an excellent singlet oxygen
generator with a UD value of 0.53. Both ZnPcC4 and ZnPcC4–BSA
exhibit efficient sonocytotoxicities against HepG2 human hepato-
carcinoma cells in the presence of ultrasound. By comparison,
ZnPcC4–BSA shows a higher sonodynamic activity with an IC50
ic effects caused by ZnPcC4–BSA (10 lM), the flow cytometry with
Annexin V-FITC and PI staining was used to distinguish early apop-
tosis from late apoptosis or necrosis [40]. As shown in Fig. 6, the
percentage of cells at early apoptotic stage for ZnPcC4–BSA treat-
ment alone was 1.6%. However, the early apoptotic cell populations
for ZnPcC4–BSA-mediated SDT increased to 11.7%. These results
show that ZnPcC4–BSA-mediated SDT could induce apoptosis in
HepG2 cells. Nevertheless, the number of cells for ZnPcC4–BSA-
mediated SDT treatment reduced, suggesting that cell death is
not only the result of apoptosis.
value of 7.5 lM. To our knowledge, this is the first report about
serum albumin conjugates of phthalocyanine as novel sonosensi-
tizers. Further development of such serum albumin conjugates
with suitable phthalocyanine may lead us to find a promising
sonosensitizer for sonodynamic therapy of cancer.
Many current studies showed that the cellular toxicity of sono-
sensitizer could be attributed to the generation of intracellular ROS
after exposure to ultrasound [7]. We also attempted to examine
whether the intracellular ROS was involved in the process of
Acknowledgments
This work was supported by the Natural Science Foundation of
China (Grant Nos. 20872016, 21172037), the Natural Science Foun-
dation of Fujian, China (Grant No. 2011J01040), and Specialized
ZnPcC4–BSA (10
lM)-mediated SDT. The intracellular ROS was
evaluated using flow cytometry with DCFH-DA as a ROS probe.
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019

H.-N. Xu et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
7
[19] W.M. Sharman, C.M. Allen, J.E. van Lier, Drug Discov. Today 4 (1999) 507.
[20] N. Yumita, S. Umemura, J. Pharm. Pharmacol. 56 (2004) 85.
[21] K. Milowska, T. Gabryelak, Ultrasound Med. Biol. 31 (2005) 1707.
Research Fund for the Doctoral Program of Higher Education
(Grant No. 20113514110001).
[22] K. Tomankova, H. Kolarova, P. Kolar, K. Kejlova, D. Jirova, Toxicol. In Vitro 23
(2009) 1465.
[23] H. Chen, X. Zhou, Y. Gao, B. Zheng, F. Tang, J.D. Huang, Drug Discov. Today 19
(2014) 502.
[24] C.F. Choi, P.T. Tsang, J.D. Huang, E.Y. Chan, W.H. Ko, W.P. Fong, D.K. Ng, Chem.
Commun. (Camb.) (2004) 2236.
[25] M.-R. Ke, J.-D. Huang, S.-M. Weng, J. Photochem. Photobiol. A 201 (2009) 23.
[26] W. Liu, T.J. Jensen, F.R. Fronczek, R.P. Hammer, K.M. Smith, M.G. Vicente, J.
Med. Chem. 48 (2005) 1033.
[27] C. Larroque, A. Pelegrin, J.E. Van Lier, Br. J. Cancer 1996 (1886) 74.
[28] W.M. Sharman, J.E. van Lier, C.M. Allen, Adv. Drug Deliv. Rev. 56 (2004) 53.
[29] X.L. Han, F.F. Tian, Y.S. Ge, F.L. Jiang, L. Lai, D.W. Li, Q.L. Yu, J. Wang, C. Lin, Y.
Liu, J. Photochem. Photobiol. B 109 (2012) 1.
[30] I. Scalise, E.N. Durantini, Bioorg. Med. Chem. 13 (2005) 3037.
[31] W. Spiller, H. Kliesch, D. Wöhrle, S. Hackbarth, B. Röder, G.J. Schnurpfeil,
Porphyrins Phthalocyanines 2 (1998) 145.
[32] M.D. Maree, N. Kuznetsova, T.J. Nyokong, J. Photochem. Photobiol. A 140
(2001) 117.
[33] J.D. Huang, P.C. Lo, Y.M. Chen, J.C. Lai, W.P. Fong, D.K. Ng, J. Inorg. Biochem. 100
(2006) 946.
[34] J. Duranteau, N.S. Chandel, A. Kulisz, Z. Shao, P.T. Schumacker, J. Biol. Chem.
273 (1998) 11619.
[35] Y. Li, T.M. Pritchett, J. Huang, M. Ke, P. Shao, W. Sun, J. Phys. Chem. A 112
(2008) 7200.
[36] Y. Liu, M.X. Xie, M. Jiang, Y.D. Wang, Spectrochim. Acta A 61 (2005) 2245.
[37] E. Alarcon, A.M. Edwards, A.M. Garcia, M. Munoz, A. Aspee, C.D. Borsarelli, E.A.
Lissi, Photochem. Photobiol. Sci. 8 (2009) 255.
[38] A. Ogunsipe, T. Nyokong, Photochem. Photobiol. Sci. 4 (2005) 510.
[39] R.P. Xiao, N. Zhang, J.D. Huang, H.H. Zhang, Guang Pu Xue Yu Guang Pu Fen Xi
31 (2011) 1052.
References
[1] H. Kolarova, K. Tomankova, R. Bajgar, P. Kolar, R. Kubinek, Ultrasound Med.
Biol. 35 (2009) 1397.
[2] N. Yumita, R. Nishigaki, K. Umemura, S. Umemura, Jpn. J. Cancer Res. 80 (1989)
219.
[3] S. Umemura, N. Yumita, R. Nishigaki, K. Umemura, Jpn. J. Cancer Res. 81 (1990)
962.
[4] D.E. Dolmans, D. Fukumura, R.K. Jain, Nat. Rev. Cancer 3 (2003) 380.
[5] Z. Huang, Technol. Cancer Res. Treat. 4 (2005) 283.
[6] S.G. Rockson, D.P. Lorenz, W.F. Cheong, K.W. Woodburn, Circulation 102 (2000)
591.
[7] I. Rosenthal, J.Z. Sostaric, P. Riesz, Ultrason. Sonochem. 11 (2004) 349.
[8] K. Tachibana, L.B. Feril Jr., Y. Ikeda-Dantsuji, Ultrasonics 48 (2008) 253.
[9] H. Shibaguchi, H. Tsuru, M. Kuroki, M. Kuroki, Anticancer Res. 31 (2011) 2425.
[10] A.H. Barati, M. Mokhtari-Dizaji, H. Mozdarani, S.Z. Bathaie, Z.M. Hassan,
Ultrasound Med. Biol. 35 (2009) 756.
[11] N. Yumita, Y. Iwase, T. Imaizumi, A. Sakurazawa, Y. Kaya, K. Nishi, T. Ikeda, S.
Umemura, F.S. Chen, Y. Momose, Anticancer Res. 33 (2013) 3145.
[12] S. Guo, X. Sun, J. Cheng, H. Xu, J. Dan, J. Shen, Q. Zhou, Y. Zhang, L. Meng, W.
Cao, Y. Tian, Int. J. Nanomed. 8 (2013) 2239.
[13] H. Tsuru, H. Shibaguchi, M. Kuroki, Y. Yamashita, M. Kuroki, Free Radical Biol.
Med. 53 (2012) 464.
[14] Z.J. Dai, S. Li, J. Gao, X.N. Xu, W.F. Lu, S. Lin, X.J. Wang, Med. Hypotheses 80
(2013) 300.
[15] X. Wang, W. Zhang, Z. Xu, Y. Luo, D. Mitchell, R.W. Moss, Integr. Cancer Ther. 8
(2009) 283.
[16] N. Yumita, S. Umemura, R. Nishigaki, In Vivo 14 (2000) 425.
[17] J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue, T.
Hasan, Chem. Rev. 110 (2010) 2795.
[40] E. Jaruga, S. Salvioli, J. Dobrucki, S. Chrul, J. Bandorowicz-Pikula, E. Sikora, C.
Franceschi, A. Cossarizza, G. Bartosz, FEBS Lett. 433 (1998) 287.
[18] J.F. Lovell, T.W. Liu, J. Chen, G. Zheng, Chem. Rev. 110 (2010) 2839.
Please cite this article in press as: H.-N. Xu et al., Preparation and sonodynamic activities of water-soluble tetra-
a-(3-carboxyphenoxyl) zinc(II) phthalo-
cyanine and its bovine serum albumin conjugate, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.019