3
62
L.-L. He et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 361–366
In the latest years, SDT has been widely investigated focusing
mol/L, was prepared in 0.05 mol/L Tris–HCl buffer solution (pH
on the antitumor effects in vivo and/or in vitro and the mecha-
nisms for the synergism between ultrasound and drugs by using
different ultrasound parameters and different sonosensitizers [6].
Most of them regarded the tumor cells as assault target, and
achieved the goal of treating tumors through inducing tumor cells
apoptosis [7–11]. However, the intracellular targets of SDT have
seldom been studied until now. The damage to intracellular sub-
stances, especially proteins that are highly abundant in cells, might
be a more effective method to kill the tumor cells [12–14]. It had
been reported that the changes of cytoskeletal F-actin had some
correlations with Ehrlich ascites carcinoma cells apoptosis, which
suggested that protein was an important subcellular target for
SDT [15]. If the proteins in the tumor cells were damaged by
sonosensitizers under ultrasonic irradiation, the whole cells would
undergo apoptosis abnormally.
7.40) containing 0.05 mol/L NaCl. MB, Diphenylcarbazide (DPCI),
c
D-Mannitol (D-Man), L-Histidine (L-His) and Ascorbic acid (V ) were
all purchased from Sinopharm Chemical Reagent Co., Ltd (China).
ꢁ4
The MB stock solution (2.00 ꢂ 10 mol/L), the DPCI stock solution
ꢁ2
(2.50 ꢂ 10 mol/L) and the different ROS scavengers stock
ꢁ
2
solution (5.00 ꢂ 10 mol/L) were all prepared in the same buffer
solution. All the other materials were of analytical reagent grade
and used without further purification. Doubly distilled water was
used to prepare solutions.
Apparatus
The fluorescence spectra were carried out on an F-7000 fluores-
cence spectrophotometer (Hitachi High-Technologies Co., Japan).
Fluorescence spectra were obtained at an excitation wavelength
of 280 nm, with the slit widths of both the excitation and emission
It has been reported that many compounds have sonodynamic
activity. Because of their widely different structure, it is difficult
to expect a universal mechanism for the synergistic effects of ultra-
sound and drugs [6]. Reviewing the probably mechanisms of SDT
which have been studied, most experimental evidence indicated
that the cell damage induced by the synergistic effects of ultra-
sound and drugs may contribute to the generation of reactive oxy-
gen species (ROS) [16–21]. ROS is a class of ubiquitous molecules
including both radicals and non-radicals such as superoxide anion
ꢁ1
set at 5.0 nm and the scanning speed of 1200 nm min . The
absorption spectra were recorded on a UV-2550 spectrophotome-
ter (Shimadzu Co., Japan) with 1.0 cm quartz cells. The Controllable
Serial-Ultrasonics apparatus (KQ5200DB, Kunshan Ultrasonic
Instruments Co., Ltd. China) shown in Fig. 2 was used as irradiation
source, operating at ultrasonic frequency of 40 kHz and output
power of 200 W through manual adjusting. All pH measurements
were made with a pHS-25 digital pH-meter (Shanghai Reaches
Instrument Co., Ltd., China).
Å
ꢁ
Å
radical ( O
2
), hydrogen peroxide (H
2
O
2
), hydroxyl radical ( OH),
1
and singlet oxygen ( O
2
). These substances are constantly formed
in the human body and have been implicated in a number of dis-
eases due to their damage to cell structures, including lipids and
membranes, proteins and nucleic acids, leading to cell death [22].
However, the ROS with strong oxidation activity can also exert
beneficial physiologic effects for many diseases, especially, for
various tumors [23].
Procedures
Effect of ultrasonic irradiation time on the damage of BSA
In two conical flasks, the final concentrations of BSA were both
ꢁ
5
1
0
.00 ꢂ 10 mol/L, and the final concentrations of MB were
Methylene blue (MB, Fig. 1) is a heterocyclic aromatic com-
pound that has antifungal, antibacterial [24] and antimalarial
activity [25]. It has been widely used to stain living organisms,
treat methemoglobinemia [26], prevent ifosfamide-induced
encephalopathy [27], and lately it has been investigated and used
as photosensitizers of PDT against several types of tumors [28].
Moreover, it has been confirmed that MB possesses sonodynamic
activities [29,30]. These results demonstrate that MB has potential
to be used as a sonosensitizer in SDT. In this work, bovine serum
albumin (BSA) was selected as a model of protein, the sonodynam-
ic damage to protein in the presence of MB was studied by fluores-
cence and absorption spectra. The mechanisms of the synergistic
effects of ultrasound and MB were studied by oxidation–extraction
photometry with several ROS scavengers. It is wished that this
report might offer some meaningful and valuable references to
promoting the application of SDT at molecule level.
ꢁ5
.00 mol/L and 1.00 ꢂ 10 mol/L, respectively. They were all
placed in the ultrasonic irradiation apparatus and the ultrasonic
irradiation time was changed from 1.0 h to 6.0 h at 1.0 h intervals.
At every time interval, the solutions (10 mL) were taken out and
detected by fluorescence spectrophotometer.
Effect of MB concentration on the damage of BSA
In six conical flasks, the final concentration of BSA were all
ꢁ
5
1
.00 ꢂ 10 mol/L, and the final concentrations of MB were chan-
ꢁ5 ꢁ5
ged from 0.00 mol/L to 2.50 ꢂ 10 mol/L at 0.50 ꢂ 10 mol/L
intervals. They were all placed in the ultrasonic irradiation appara-
tus for 3.0 h. Then, the solutions were taken out and detected the
fluorescence spectra.
Experimental section
Materials
BSA (Fraction V) was obtained from Amresco (USA) and used
without further purification. The BSA stock solution, 2.50 ꢂ 10ꢁ
5
N
CH3
H C
3
N
S
N
Cl-
CH3
CH3
Fig. 1. Molecular structure of MB.
Fig. 2. The apparatus of ultrasonic irradiation.