Ultrasound-dependent cytoplasmic internalization of a peptide-sonosensitizer conjugate

Article abstract of DOI:10.1016/j.bmc.2017.06.024

A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting

Full text of DOI:10.1016/j.bmc.2017.06.024

Accepted Manuscript
Ultrasound-dependent cytoplasmic internalization of a peptide-sonosensitizer
conjugate
Yuki Inaba, Kazunori Watanabe, Mizuki Kitamatsu, Eiji Nakata, Atsushi
Harada, Takashi Ohtsuki
PII:
S0968-0896(17)30582-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.024
BMC 13807
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 March 2017
6 June 2017
13 June 2017
Please cite this article as: Inaba, Y., Watanabe, K., Kitamatsu, M., Nakata, E., Harada, A., Ohtsuki, T., Ultrasound-
dependent cytoplasmic internalization of a peptide-sonosensitizer conjugate, Bioorganic & Medicinal Chemistry
(2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.024
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Bioorganic & Medicinal Chemistry
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Ultrasound-dependent cytoplasmic internalization of a peptide-sonosensitizer
conjugate
Yuki Inabaa, Kazunori Watanabea, Mizuki Kitamatsub, Eiji Nakatac, Atsushi Haradad, Takashi Ohtsukia,*
aDepartment of Medical Bioengineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan
bDepartment of Applied Chemistry, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
cInstitute of Advanced Energy, Kyoto University, Gokasho,Uji, Kyoto 611-0011, Japan
dDepartment of Applied Chemistry, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, Japan
*Corresponding author. E-mail: ohtsuk@okayama-u.ac.jp (T. Ohtsuki)
ARTICLE INFO
ABSTRACT
Article history:
Received
A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a
molecular conjugate of a cell-penetrating peptide (CPP),
a functional peptide, and a
Received in revised form
Accepted
Available online
sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat
CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized.
CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused
molecules tend to become entrapped in endosomes, as was observed for TatBim-RB
molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells
were irradiated with ultrasound, which successfully induced endosomal escape and
cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that
this peptide-sonosensitizer conjugate strategy may facilitate numerous kinds of medicinal
chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel
therapeutic agent for the promotion of apoptosis.
Keywords:
Sonosensitizer
Ultrasound
Cell-penetrating peptide
Endosomal escape
Apoptosis
1. Introduction
extensive evaluations of US for medical purposes.14,15 Recently,
high-intensity, focused ultrasound has attracted attention because
Recently, the fusion of peptides and proteins to cell-
penetrating peptides (CPPs) to facilitate their transport into cells
has been widely used for therapeutic and biological purposes.1,2
However, this strategy is often limited by the inefficient transfer
of the CPP-fused peptides and proteins to the cytosol consequent
to their endosomal entrapment.3 One of the methods to overcome
this problem is to use photosensitizers and light to mediate
endosomal escape.4,5 In this method, the light causes the
entrapped photosensitizer to generate reactive oxygen species
(ROS), which disrupts the surrounding endosomal membrane.
For example, we previously designed a TatBim-Alexa molecule,6
comprising a conjugate of Tat CPP from the HIV-1 transactivator
of transcription (TAT) protein,7 the BH3 domain derived from
Bim apoptosis-inducing protein,8,9 and the Alexa Fluor 546 dye
as a photosensitizer. TatBim-Alexa molecules enter cells by the
endocytic pathway, are entrapped in endosomes, and then escape
from the endosomes and induce apoptosis by photoirradiation.
Similar fusion molecules, such as TatU1A-Alexa and TatU1A-
DY750 for photo-dependent cytoplasmic RNA delivery, were
also reported.10–13 However, the poor tissue penetration of light
limits the application of these photosensitizing molecules.
Alternatively, ultrasound (US) represents a promising substitute
for light as an external stimulus because of its deeper penetrating
property. The high tissue-penetrating ability has prompted
it can specifically irradiate a target tissue and provides a potential
noninvasive therapeutic strategy.16
Sonosensitizers are known as molecules that generate ROS in
a
US-dependent manner. Sonosensitizers include inorganic
materials (e.g. titanium dioxide)17 and organic dyes (e.g.
porphyrin derivatives and rose bengal).18–20 In the current study, a
sonosensitizer- and CPP-fused functional peptide was developed
as a molecule exhibiting US-dependent intracellular function. As
an example of this design, TatBim-RB, containing the Tat CPP,
the Bim apoptosis-inducing peptide, and the sonosensitizer rose
bengal was synthesized. The use of a sonosensitizer and US
instead of a photosensitizer and light was attempted to facilitate
endosomal escape of the CPP-fused functional peptide (Fig. 1).

Figure 2. Phase contrast and TatBim-RB fluorescence images immediately
after US irradiation. Cellular images in the middle of the irradiated area are
shown. TatBim-RB was imaged by rose bengal fluorescence. CHO cells were
treated with TatBim-RB and irradiated with 1 or 3 MHz pulsed US (30% duty
cycle) at 0.5 W/cm2 for 15 min. Scale bars indicate 100 µm.
Figure 1. Conceptual diagram of US-dependent cytoplasmic internalization
of TatBim-RB.
2. Results and Discussion
2.2. Detection of apoptosis after the treatment with TatBim-RB
and US
2.1. US-dependent internalization of TatBim-RB
TatBim-RB was synthesized by reacting TatBim bearing a
Cys residue at the C-terminus with rose bengal maleimide. After
the purification, the majority of free rose bengal maleimide was
removed from TatBim-RB (Fig. S1 in Supporting Information).
To attempt US-dependent internalization of TatBim-RB, Chinese
hamster ovary (CHO) cells were treated with 2 µM TatBim-RB
followed by US as described in Materials and Methods (Fig. 2).
Without US irradiation, low levels of TatBim-RB were detected
in the cells with a dotted localization pattern, indicating that the
TatBim-RB was entrapped in endocytotic compartments as
previously shown using a similar Tat-fused molecule.11 Tat-fused
peptide and protein tends to enter cells by macropinocytosis.21,22
After US irradiation, stronger and diffuse fluorescence of
TatBim-RB was observed in the cells, indicating that TatBim-RB
molecules had escaped from the endocytotic compartments and
diffused into the cytosol. The increase of TatBim-RB
fluorescence was probably due to the release from the
concentration quenching and the pH increase upon relocalization
from endosomes to the cytoplasm, as the rose bengal
fluorescence ratio (pH 7.6/ pH 5.2) was 1.4. Both of 1 MHz and
3 MHz US induced cytoplasmic internalization of TatBim-RB by
the cells. These frequencies are included in the common
frequency range of ultrasonography, which has already been
applied in the clinic. In general, the depth of penetration of US
depends on the frequency.23 For example, the penetration
property of 10 MHz is 3.5-fold higher than that of 20 MHz.
Hence, we chose a lower frequency (1 MHz) for the following
experiments. Maximum tissue penetration ability of 1 MHz US
was reported to be about 80 cm.24
Next, we evaluated apoptosis induced by TatBim-RB and US
(Fig. 3). CHO cells were treated with TatBim-RB and irradiated
with US. At 8 h after the irradiation, apoptotic cells were stained
with NucView488. Apoptosis was detected in the cells treated
with TatBim-RB followed by US but not in the cells treated with
TatBim-RB only. Therefore, TatBim-RB at this concentration (2
µM) induced apoptosis US-dependently. TatBim-RB images
indicated the US-dependent cytoplasmic dispersion of TatBim-
RB, as observed in Figure 2, although the timing of the imaging
differed, with the images in Figure 2 being obtained immediately
after the irradiation whereas those in Figure 3 were obtained at 8
h after the irradiation. US did not induce apoptosis in the absence
of TatBim-RB or in the presence of TatBim peptide without
conjugated rose bengal. In addition, we investigated the effects of
US intensity toward the cellular internalization of TatBim-RB
(Fig. S2 in Supporting Information). As expected, US at stronger
irradiation induced a higher efficiency of apoptosis. Direct
cytotoxic effect of TatBim-RB/US-induced ROS, independent of
Bim activity, was estimated by trypan blue staining, which
suggested that this effect was not apparently shown (Fig. S3 in
Supporting Information). Furthermore, as a previous report
showed that cell viability was decreased by US irradiation in the
presence of 100 µM rose bengal but not by US in the presence of
10 µM rose bengal,25 we expected that the Bim-independent
damage was not likely to be apparent in our experimental
conditions using 2 µM TatBim-RB but may appear in conditions
using higher TatBim-RB concentrations.

with the results of the cellular TatBim-RB internalization
experiments (Fig. S5 in Supporting Information). Specifically,
US-dependent TatBim-RB internalization and apoptosis were not
observed in a polyethylene 96-well culture plate whereas both
were observed when a glass-bottomed dish was used and the cell-
culture solution was applied only on the glass region.
Figure 4. US-induced generation of ROS in the presence of rose bengal. ROS
was detected using the fluorescent ROS indicator H2DCF on a glass-bottomed
dish (a) and in a 96-well plate (b).
Figure 3. Apoptosis induction in CHO cells. Cellular images were captured
at 8 h after US irradiation (1 MHz, 0.5 W/cm2, duty cycle 30%, 15 min).
Apoptosis was detected using a NucView 488 Caspase-3 Assay Kit. To avoid
photochemical damage of the cells by rose bengal excitation, TatBim-RB
images were obtained after the apoptosis imaging. Scale bars indicate 100
µm.
2.4. Contribution of sonoluminescence to US-induced ROS
generation
Rose bengal has been shown to act as a sonosensitizer;25
however, its sonosensitizing mechanism has not been clarified.
One possible explanation is that sonoluminescence19,28 generated
by the US energy might cause excitation of rose bengal and
initiate a photochemical process resulting in ROS generation. To
address this possibility, sonoluminescence was measured and its
intensity was compared to the light intensity necessary for
photochemical internalization of TatBim-RB by cells. In this
measurement, the rose bengal concentration and US conditions
were similar to the previous cellular experiments. Figure S6 in
the Supporting Information shows that sonoluminescence could
barely be detected, representing less than 1/1000 of the light
conditions for a typical photochemical internalization, indicating
that sonoluminescence does not constitute the main pathway for
the US-dependent rose bengal excitation followed by ROS
generation. This observation agrees with the results that singlet
oxygen was also barely detected in US-irradiated rose bengal
solution (Fig. S4 in the Supporting Information), because singlet
oxygen would be generated if rose bengal is excited by light.29
2.3. Mechanism of US-independent TatBim-RB endosomal
escape
A fundamental question related to this study is the means by
which US induces the endosomal escape of TatBim-RB. A
minimal answer may be that US induces ROS generation in the
presence of TatBim-RB and the endosomal membrane might be
disrupted by the attack of ROS generated from the endosomally
entrapped TatBim-RB. We confirmed the US-dependent
generation of ROS in the presence of rose bengal using 2’, 7’-
dichlorodihydrofluorescein (H2DCF) (Fig. 4a); additionally, a
previous report has also shown the same phenomenon using 1,3-
diphenylisobenzofuran.25 US-induced ROS was hardly generated
in the presence of TatBim peptide (data not shown). In a
photochemical internalization strategy using photosensitizers and
light, photoinduced singlet oxygen (1O2) has been reported as a
main trigger for endosomal escape4,26. Therefore, we evaluated
US-induced 1O2 generation using singlet oxygen sensor green
(SOSG) (Molecular Probes, Eugene, OR, USA); however, rose
3. Conclusions
This study aimed to develop an US-dependent cytosolic
peptide delivery method. The US-sensitive molecule was
designed such that a functional peptide of interest was conjugated
with a CPP and a sonosensitizer. TatBim-RB was prepared as a
conceptual model of the sonosensitizer- and CPP-fused
functional peptide. TatBim-RB entered cells but was entrapped in
endocytotic compartments; its escape from the compartments
was subsequently facilitated by US irradiation. Thus, US
treatment induced cytoplasmic TatBim-RB delivery and
subsequent apoptosis. This peptide delivery method is novel,
differing from the other primary US-dependent method,
sonoporation, which utilizes nano- and microbubbles.30
Sonosensitizers have been used in the study of sonodynamic
therapy (SDT) for cancer treatments and are known to induce
ROS-mediated cytotoxicity under US irradiation.31 Our US
irradiation conditions are not stronger than those of most SDT
studies,31 and much weaker than high-intensity focused
1
bengal-dependent O2 generation was barely detected under US
irradiation (Fig. S4 in Supporting Information). This indicated
that US with rose bengal induced the generation of ROS that
were reactive with H2DCF, although the generated ROS were
mainly of types other than 1O2.
It should be noted that US-induced ROS generation was
dependent on the materials used for cell culture. US-induced
ROS generation in a polyethylene 96-well culture plate was
much less than that on a glass-bottomed dish (Fig. 4). The
difference of the US-induced ROS generation between these
substrates appeared to be due to differences of the reflection
coefficients of the water-polyethylene (7.7%) and water-glass
(80%) interfaces, which were estimated using an established
equation.27 This difference of ROS generation was coordinate

ultrasound (HIFU) treatments.32 The present peptide-delivery
method also utilized a sonosensitizer but is distinct from the
simple cell disruption method. Furthermore, a previous report
demonstrated that cell viability was decreased by US irradiation
in the presence of 100 µM rose bengal but not affected by US in
the presence of 10 µM rose bengal.25 In the current study, the
cells were treated with 2 µM TatBim-RB; thus, the direct
cytotoxic effect of TatBim-RB/US-induced ROS, independent of
Bim activity, was not high. Therefore, the present method is
considered promising for in vivo biological studies, as it allows
the delivery of functional peptides into target cells and may
represent an effective alternative to conventional SDT.
with NucView 488 were observed using an IX51 fluorescence
microscope (λex = 470–490 nm).
CHO cells were treated with 2 µM TatBim-RB and irradiated
with US, as described above. After US-irradiation followed by
two washes with T buffer, the cells were incubated with Ham’s
F12/10% FBS medium at 37 °C for 8 h. Then, the buffer on the
cells was removed and the cells were stained using the NucView
488 Caspase-3 assay kit (Thermo Fisher Scientific) in T buffer at
37 °C for 30 min. After the staining, the cells were washed with
T buffer and the apoptotic cells stained with NucView 488 were
observed using the IX51 fluorescence microscope (λex = 470–
490 nm).
4. Materials and methods
4.1. Preparation of TatBim-RB
To covalently attach rose bengal to peptides, rose bengal
maleimide, in which the maleimide group can react with the thiol
group of peptides, was synthesized as described.26 The TatBim-C
peptide (H-RKKRR QRRRE IWAQE LRRIG DEFNA
YYARGC-NH2) was prepared by conventional Fmoc-based
solid-phase peptide synthesis. TatBim-C containing a cysteine at
the C-terminus was reacted with rose bengal-maleimide to
generate TatBim-RB. The reaction mixture containing 10 mM
HEPES-KOH (pH 7.6), 2 µM Tris (2-carboxyethyl) phosphine,
25 µM TatBim-C, and 25 µM rose bengal-maleimide was
incubated at room temperature for 1 h. TatBim-RB was purified
by reversed-phase HPLC (Symphonia C18 Column [4.6 × 150
mm, 5 µm particle diameter, Jasco, Tokyo, Japan]) eluted with
0.1% aqueous trifluoroacetic acid (A)/acetonitrile (B) gradient
mixture (B: 0 min; 0%, 10 min; 40%, 30 min; 65%, 40 min;
100%) at a flow rate of 0.6 mL/min. The purified TatBim-RB
was analyzed by SDS-PAGE. Rose bengal fluorescence in the gel
was imaged using an FLA-9000 imager (Fujifilm, Tokyo, Japan)
with λex= 532 nm.
Figure 5. Setup for irradiation of the cells with US. The cells were cultured
and adhered on the glass region of a glass-bottomed dish (glass diameter 12
mm). The cell-culture solution (200 µL) remained in contact only with the
glass region by utilizing the surface tension. The US probe (ϕ 6-mm) of the
Sonitron2000V apparatus was placed above the center of the dish. The probe
tip was placed in contact with the surface of the cell-culture solution and the
cells were irradiated.
4.4. Detection of ROS
4.2. US-dependent internalization of TatBim-RB
Production of ROS was detected using 2′, 7′-
dichlorodihydrofluorescein diacetate (H2DCF-DA) (Wako).
H2DCF-DA (10 mM) was first treated with NaOH (10 mM) for
30 min at room temperature to generate H2DCF, which is a ROS
indicator that can be rapidly oxidized to generate highly
fluorescent 2′, 7′- dichlorodihydrofluorescein. H2DCF (10 µM)
and rose bengal (10 µM) were mixed in 100 µL T buffer and
loaded on a MicroWell 96-well optical bottom plate (Nunc,
Rochester, NY, USA) or on a glass (12-mm)-bottomed dish
(IWAKI). The solution was irradiated with US (1 MHz, duty
cycle 30%, 0.3 W/cm2) using a Sonitron2000V equipped with an
US probe (ϕ 6-mm). After irradiation, fluorescence spectra were
measured at an excitation wavelength of 492 nm using an FP-
6600 spectrofluorometer (Jasco).
CHO cells were cultured at 37 °C under 5% CO2 in Ham’s
F12 medium (Wako Pure Chemical Industries, Osaka, Japan)
supplemented with 10% fetal bovine serum (FBS) (Sigma, St.
Louis, MO, USA), 100 units/mL penicillin (Gibco, Gaithersburg,
MD, USA), and 100 µg/mL streptomycin (Gibco). Confluent
CHO cells (70–90%) were incubated in a 12-mm glass-bottomed
dish (IWAKI, Japan) for 2 h at 37 °C with 2 µM TatBim-RB in T
buffer (20 mM HEPES-KOH (pH 7.4), 115 mM NaCl, 5.4 mM
KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 13.8 mM glucose)
under an atmosphere of 5% CO2. After washing the cells twice
with T buffer, the cells in T buffer were irradiated with
unfocused US using a Sonitron2000V equipped with an US
probe (ϕ 6 mm) (Nepa Gene, Ichikawa, Japan). The US probe
was set so that the probe tip touched the surface of the cell-
culture solution (Fig. 5). After the irradiation, intracellular
internalization of TatBim-RB was visualized using an IX51
fluorescence microscope with a DP72 digital camera (Olympus,
Japan).
Acknowledgments
We thank Shota Nagahiro (Okayama University) for help with the
revision. This work was supported by JSPS KAKENHI (No.
25560224 to T. O., A. H., and E. N.), Grants-in-Aid for Scientific
Research on Innovative Areas "Nanomedicine Molecular Science"
(26107711 to T. O.), and the “Joint Usage/Research Program on
Zero-Emission Energy Research”, Institute of Advanced Energy,
Kyoto University (ZE28B-32).
4.3. Detection of apoptosis following treatment with TatBim-RB
and US
CHO cells were treated with 2 µM TatBim-RB and irradiated
with US, as described above. After US-irradiation followed by
two washes with T buffer, the cells were incubated with Ham’s
F12/10% FBS medium at 37 °C for 8 h. Then, the buffer on the
cells was removed and the cells were stained using a NucView
488 Caspase-3 assay kit (Thermo Fisher Scientific, Waltham,
MA, USA) in T buffer at 37 °C for 30 min. After the staining, the
cells were washed with T buffer and the apoptotic cells stained
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Graphical Abstract