ARTICLE IN PRESS

Ultrasound in Med. & Biol., Vol. ■■, No. ■■, pp. ■■–■■, 2018
Copyright © 2018 World Federation for Ultrasound in Medicine & Biology. All rights reserved.
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https://doi.org/10.1016/j.ultrasmedbio.2018.02.003

● Original Contribution

PRE-TREATMENT WITH EITHER L-CARNITINE OR PIRACETAM INCREASES

ULTRASOUND-MEDIATED GENE TRANSFECTION BY REDUCING
SONOPORATION-ASSOCIATED APOPTOSIS
Wei-Hao Liao,*,† Chueh-Hung Wu,*,‡ and Wen-Shiang Chen*,§
* Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, Taipei, Taiwan; † National Taiwan
University College of Medicine, Taipei, Taiwan; ‡ Institute of Biomedical Engineering, College of Medicine and College of
Engineering, National Taiwan University, Taipei, Taiwan; and § Division of Medical Engineering Research, National Health
Research Institutes, Miaoli, Taiwan
(Received 3 July 2017; revised 2 February 2018; in final form 3 February 2018)

Abstract—Sonoporation, the use of ultrasound to alter the permeability of cell membranes, is a non-viral tech-
nique used to facilitate gene delivery, possibly by opening transient pores in the cell membrane. However, sonoporation
may have negative bio-effects on cells, such as causing apoptosis, which limits its efficacy in gene delivery. In this
study, we investigated whether pre-treatment with either L-carnitine or piracetam could protect cells from un-
dergoing apoptosis after sonoporation and the possible mechanisms. We found that either L-carnitine or piracetam
can promote gene transfection without reducing cell viability, possibly by reducing cavitation-induced reactive
oxygen species generation, reversing alterations of mitochondrial membrane potential, preventing caspase-3/7 ac-
tivity and facilitating mitochondrial ATP production. In conclusion, pre-treatment with either L-carnitine or piracetam
could protect cells from sonoporation-associated apoptosis by preserving mitochondrial function.
(E-mail: b88401062@ntu.edu.tw) © 2018 World Federation for Ultrasound in Medicine & Biology. All rights
reserved.
Key Words: Carnitine, Piracetam, Ultrasound, Gene, Apoptosis.

INTRODUCTION disruption of microbubbles exposed to ultrasound, pro-

motes the transfer of extracellular materials into cells by
Gene therapy involves the delivery of genetic material into
opening transient pores in the cell membrane (Lentacker
cells to provide an effective treatment for various dis-
et al. 2014). However, it may have negative bio-effects on
eases such as cancer and congenital diseases. For successful
cells, such as cell destruction (Hassan et al. 2010), free
gene therapy, it is necessary to develop techniques that can
radical formation (Honda et al. 2004), cell cycle arrest
deliver therapeutic genes to tissues or cells efficiently, se-
(Chen et al. 2013; Zhong et al. 2011), necrosis (Hassan
lectively and safely. In recent years, ultrasound (US)
et al. 2010) and apoptosis (Honda et al. 2004; Hutcheson
combined with microbubbles has been reported to be a non-
et al. 2010; Miller and Dou 2009; Zhong et al. 2011, 2013).
viral method for delivering genes in vitro and in vivo
Previous studies have indicated that sonoporation can
(Lentacker et al. 2014; Li et al. 2009; Rychak and Klibanov
perturb endoplasmic reticulum (ER) homeostasis, cause
2014; Tomizawa et al. 2013). US-mediated delivery (also
mitochondria to lose their membrane potential (Δψm), in-
called sonoporation) has low immunogenicity and inva-
crease mitochondria-activated apoptosis initiators (caspase
siveness, site specificity, localization and high penetration
family members) and finally result in the initiation of apop-
and safety for repeated treatments compared with other
tosis (Honda et al. 2004; Zhong et al. 2013). Therefore,
delivery techniques (Delalande et al. 2013; Rychak and
protection of cells from the negative effects of sonoporation
Klibanov 2014). Sonoporation, which is induced by the
is an important issue when using sonoporation for ther-
apeutic purposes.
To protect mitochondria in particular from the neg-
Address correspondence to: Chueh-Hung Wu, Departments of Physical ative bio-effects of sonoporation, L-carnitine and piracetam
Medicine and Rehabilitation, National Taiwan University Hospital, College
of Medicine, National Taiwan University, Taipei, Taiwan. E-mail: may be feasible candidates as they have been reported to
b88401062@ntu.edu.tw protect cells from undergoing apoptosis through the

1
 ARTICLE IN PRESS
2 Ultrasound in Medicine & Biology
preservation of mitochondrial function (Chang et al. 2005;
Costa et al. 2013; Keil et al. 2006; Kurz et al. 2010; Leuner
et al. 2010; Moosavi et al. 2016; Stockburger et al. 2013;
Ye et al. 2010). L-Carnitine could protect cells from various
stresses through the reduction of oxidative injury, mito-
chondrial dysfunction, caspase activation and, finally,
inhibition of apoptosis (Chang et al. 2005; Costa et al. 2013;
Moosavi et al. 2016; Ye et al. 2010). Furthermore,
L-carnitine significantly increased the activity of endog-
enous antioxidant enzymes (Ye et al. 2010) and ATP
production (Bremer 1983) and attenuated aging (Arockia
Rani and Panneerselvam 2001). With respect to piracetam,
it is a nootropic drug commonly used in a clinical context,
and is used to improve cognitive impairment in aging, brain
injuries, Alzheimer’s disease and ischemia (Keil et al. 2006;
Leuner et al. 2010; Stockburger et al. 2013). Piracetam
has been found to improve mitochondrial membrane po-
tential and the impairment of mitochondrial function and
apoptosis after oxidative stress (Costa et al. 2013; Keil et al.
2006; Kurz et al. 2010; Leuner et al. 2010; Stockburger
et al. 2013). Furthermore, the combination of L-carnitine
and piracetam has additive effects on the protection against
PC3 cell necrosis induced by simvastatin (Costa et al. 2013).
Although L-carnitine and piracetam have been reported
to protect mitochondrial function in different circum-
stances, their roles in mitochondrial protection from
sonoporation have not been investigated. Therefore, we
aimed to test whether L-carnitine or piracetam could protect
cells against the negative bio-effects induced by
sonoporation.
METHODS
Cell culture and plasmid preparation
NIH3T3 cells (from Bioresource Collection and
Research Center, Taiwan), an immortalized mouse
fibroblast cell line, were maintained in Dulbecco’s modi-
fied Eagle’s medium containing 4.5 g/L glucose (DMEM,
high glucose, Invitrogen, USA), supplemented with
10% bovine calf serum (Invitrogen, USA), and a 1 × an-
tibiotic–antimycotic (Invitrogen, USA) at 37 °C in 5%
CO2.
BNL CL.2 (from Bioresource Collection and Re-
search Center, Taiwan) is a normal embryonic liver cell
line derived from a BALB/c mouse. BNL cells were main-
tained in high-glucose DMEM supplemented with 10%
fetal bovine serum (Invitrogen, USA) and a 1 × antibiotic–
antimycotic at 37 °C in 5% CO2.
Firefly luciferase gene (luc, Promega, Madison, WI,
USA) was subcloned into the pCI-neo mammalian ex-
pression vector (Promega) and called pCI-neo-luc. Plasmid
DNA was prepared using the EndoFree Plasmid Mega kit
(Qiagen, Valencia, CA, USA) according to the manufac-
turer’s instructions.
Volume ■■, Number ■■, 2018
US exposure
The US field was generated by a 1-MHz plane piezo-
electric transducer with an active diameter of 3.8 cm (A392
S, Panametrics, Waltham, MA, USA). A 24-well plate was
placed 5.4 cm above the transducer, and only one well at
a time was exposed to US. We chose the 5.4-cm distance
to ensure that the culture well was within the −3-dB pres-
sure distribution in the near field of the transducer. The
volume inside the exposure well was 2 mL. The duty cycle
was set to 20%. The pulse length was fixed at 27.5 ms for
exposure conditions. A needle hydrophone (SPEH-S-
0500, ONDA, Sunnyvale, CA, USA) was located near the
sonicated well and used to detect the cavitation noise in
the medium. The received signal was amplified, digi-
tized, transferred to a computer and analyzed with the
Labview program. The cavitation activity was quantified
using an inertial cavitation index (CI). The designed cavi-
tation regulation system was based on the instantaneous
calculation of the inertial cavitation level. This feedback
loop process imposes a precise cavitation level for pro-
viding better control of cavitation. Further details of the
acquisition device, calculation of CI and feedback control
system can be found in previous reports (Lo et al. 2014;
Sabraoui et al. 2011).
Cultured NTH3 T3 cells or BNL cells were initially
plated in a 24-well plate at a density of 105 cells/well for
18 h. The medium was replaced with 2 mL of fresh
medium, and cells were treated with the indicated doses
of L-carnitine (Sigma, St. Louis, MO, USA) or piracetam
(Sigma) for 4 h before US exposure. The medium was then
immediately supplemented with 10 µg of pCI-neo-luc
plasmid and 5 µl of SonoVue contrast agent (Bracco, Milan,
Italy). The cells were exposed to US bursts with various
CIs (CI = 10, 15) for 110 s.
At various post-exposure points (2 and 18 h), the cells
were harvested for further analysis, as described in the fol-
lowing sections. The 18-h point was chosen as it takes time
for gene expression to produce sufficient proteins for anal-
ysis. It also takes time for cell culture to test viability and
caspase activity. On the other hand, the membrane poten-
tial alteration, ROS generation and ATP generation induced
by US may return to baseline in a short time and cannot
be detected after 18 h. Cell proliferation after18 h of in-
cubation may also interfere with ATP detection. Therefore,
we analyzed membrane potential alteration, ROS gener-
ation and ATP generation in 2 h.
Cell viability assay
Cell viability on US exposure was measured using
the alamarBlue assay (Thermo Fisher Scientific, USA).
After 18 h of incubation, NIH3T3 cells were washed twice
with phosphate-buffered saline solution and incubated with
10% v/v alamarBlue medium. The plate was incubated
under standard conditions of 37 °C and 5% CO2 for 30 min,
 ARTICLE IN PRESS
Protecting against sonoporation-induced bio-effects ● W.-H. Liao et al.
and fluorescence measurements were performed with a
microplate reader (Infinite M200, Tecan, Austria). Rela-
tive cell viability was calculated as the ratio of relative
fluorescence unit (RFU) to that upon mock treatment
(RFUsample/RFUmock) × 100%. Mock experiments were
treated with 10 µg of pCI-neo-luc plasmid and 5 µL of
SonoVue contrast agent, with the drug substituted with an
equivalent volume of ddH2O. All experiments were per-
formed in triplicate.
Gene transfection evaluation
The efficiency of transfection was evaluated by the
luciferase assay system (Promega, Madison, WI, USA),
as described in the manufacturer’s protocol. After US ex-
posure and another 18-h incubation, cells were washed by
PBS twice and then lysated by cell culture lysis reagent
(Promega, WI, Madison, USA). Samples were then cen-
trifuged at 14,000 rpm and 4 °C for 10 min to remove the
cell debris. Supernatant was measured by a microplate
luminometer (Infinite M200, Tecan, Austria). Luciferase
activity is represented as relative light units (RLU) per mil-
ligram of protein. Protein concentration was measured using
the Bio-Rad protein assay (Bio-Rad Laboratories, Her-
cules, CA, USA). All experiments were performed in
triplicate.
Measurement of cell apoptosis
For quantitative analysis of apoptosis, apoptotic cells
were detected using the eBioscience Annexin V–fluorescein
isothiocyanate (FITC) apoptosis detection kit (Invitrogen,
USA) according to the manufacturer’s instructions. After
US exposure and another 18 h of incubation, cells were
trypsinized, washed once in binding buffer and then in-
cubated with Annexin V-FITC/propidium iodide (PI) in
binding buffer at room temperature. After 15 min of in-
cubation, the cells were immediately analyzed with LSRII
flow cytometry (BD Biosciences, San Jose, CA, USA) and
FlowJo software (Treestar Software, San Carlos, CA, USA).
Early apoptotic cells were represented as Annexin V-FITC
positive and PI negative. All experiments were per-
formed in triplicate.
Mitochondrial membrane potential assay
Mitochondrial membrane potential (Δψm) was ana-
lyzed using JC-1 dye (Invitrogen, USA). After US exposure
and another 2 h of incubation, the treated cells were in-
cubated with 10 µg/mL JC-1 for 10 min at 37 °C to monitor
mitochondrial membrane potential. JC-1-stained cells were
then analyzed by LSRII flow cytometry (BD Biosci-
ences, San Jose, CA, USA) and analyzed using FlowJo
software. All experiments were performed in triplicate.
3
Caspase-3/7 activity assay
Caspase-3/7 activity was measured using the Caspase-
Glo Caspase-3/7 assay (Promega), as described in the
manufacturer’s protocol. At 18 h post-exposure, NIH3T3
cells were harvested and seeded in a 96-well plate at
1.2 × 104 cells/well. The cells were then immediately mixed
with 100 µL of equilibrated Caspase-Glo 3/7 reagents and
incubated for 10 min at room temperature. After incuba-
tion, caspase activity was measured with a microplate reader
(Infinite M200, Tecan). Caspase activity indicates the RLU
per milligram of protein. Protein concentration was mea-
sured using the Bio-Rad protein assay. All experiments were
performed in triplicate.
Measurement of accumulation of reactive oxygen
species
After US exposure and another 2 h of incubation, to
monitor the accumulation of reactive oxygen species (ROS),
the treated cells were incubated with 5 µM 2’,7’-
dichlorodihydrofluorescein diacetate (DCF-DA) for 30 min
at 37 °C. Cells were subsequently washed twice and har-
vested, and the percentage of DCF-positive cells was
quantified using LSRII flow cytometry and analyzed using
FlowJo software. All experiments were performed in
triplicate.
Luminescent ATP detection assay
Cellular ATP levels were determined using the ATP
determination kit (Abcam, UK), in accordance with the
manufacturer’s instructions. After US exposure and another
2 h of incubation, cells were washed and lysed using
100 µL of detergent buffer. The lysates were then centri-
fuged at 14,000 rpm and 4 °C for 10 min to pellet the cell
debris. The protein concentration of the lysates was de-
termined as described before, triplicates of 40 µL of lysates
were used for ATP determination and luminescence was
measured using a microplate reader (Infinite M200, Tecan).
Relative ATP indicates RLU per milligram of protein. All
experiments were performed in triplicate.
Statistics
All numerical data are presented as the mean ± stan-
dard deviation. Tests of significance were performed using
one-way analysis of variance with a post hoc Tukey test.
A p value < 0.05 was considered to indicate statistical sig-
nificance and is denoted by an asterisk in the figures.
RESULTS
L-carnitine and piracetam promote gene transfection
without reducing cell viability after sonoporation
Pre-treatment of NIH3T3 cells with 20 µM L-carni-
tine or 20 µM piracetam efficiently increased US-mediated
gene transfection compared with that of untreated cells
 ARTICLE IN PRESS
4 Ultrasound in Medicine & Biology
Fig. 1. Effect of L-carnitine or piracetam on ultrasound (US)-
mediated transfection. NIH3T3 cells were pre-treated with two
concentrations of L-carnitine or piracetam and then transfected with
US (1 MHz, cavitation index = 10, 110 s). After 18 h of incuba-
tion, luciferase activity (a) and cell viability (b) were analyzed.
Luciferase activity is represented as relative luminescence units (RLU)
per milligram of protein. There was no luciferase activity in all groups
not exposed to US treatment. *p < 0.05.
(Fig. 1a). Pre-treatment with 10 µM L-carnitine or 10 µM
piracetam also increased gene transfection compared with
that of untreated cells, but to a lesser degree. As for the
cytoprotective effects against US-associated apoptosis, the
cell survival rate did not decrease significantly after
sonoporation on pre-treatment with L-carnitine or
piracetam, but did decrease significantly if the cells were
left untreated (Fig. 1b). Pre-treatment with L-carnitine or
piracetam promoted US-mediated gene transfection without
negatively affecting cell viability.
Increasing US intensity promoted luciferase gene
transfection efficiency accordingly. Pre-treatment with
L-carnitine or piracetam further increased US-induced lu-
ciferase gene expression at various US intensities in
NIH3T3 cells (CI = 10 or 15) (Fig. 2a). Similar results were
obtained in the BNL cell line I which pre-treatment with
Volume ■■, Number ■■, 2018
either L-carnitine or piracetam increased US-mediated gene
transfection (Fig. 2b). The cell viability assay indicated
that pre-treatment of cells with L-carnitine or piracetam
protected them against death after sonoporation (Fig. 2c).
These results suggest that L-carnitine or piracetam
reduced the negative impacts of US. Because US inten-
sity at CI = 15 offered the best transfection efficiency, it
was used for subsequent experiments.
L-carnitine and piracetam alleviate US-induced
apoptosis
To evaluate the direct effect of L-carnitine or piracetam
on US-induced apoptosis, we observed apoptotic cells by
annexin V-FITC and propidium iodide (PI) staining (Fig. 3a,
3b). Pre-treatment of NIH3T3 cells with 20 µM L-
carnitine or piracetam predominantly reduced US-mediated
apoptotic cells compared with untreated controls. These
results indicate that sonoporation-induced apoptosis could
be inhibited by pre-treatment with L-carnitine and
piracetam.
L-carnitine and piracetam reduce sonoporation-induced
ROS generation in NIH3T3 cells
To evaluate the direct effect of L-carnitine or piracetam
on US-induced oxidative stress, we measured ROS for-
mation by DCF-DA staining. US exposure significantly
increased the intracellular ROS level, whereas pre-
treatment of cells with L-carnitine or piracetam decreased
US-associated ROS production (Fig. 4). These findings sug-
gested that L-carnitine or piracetam may alleviate apoptosis
that has been induced by US by reducing intracellular ROS
generation.
US-induced alterations of mitochondrial membrane
potential (δψm) are reversed by L-carnitine and
piracetam
A previous study indicated that loss of Δψm is induced
by sonoporation (Zhong et al. 2013). NIH3T3 cells exposed
to US exhibited a significant increase in fluorescence in-
tensity compared with non-exposed cells, indicating that
US exposure was associated the loss of Δψm in NIH3T3
cells (Fig. 5). Pretreatment with L-carnitine or piracetam
significantly ameliorated mitochondrial membrane damage
and its potential collapse associated with sonoporation.
Among five conditions under US treatment, a signifi-
cantly lower percentage of cells with depolarized
mitochondria was observed in 20 µM piracetam than in
the control (p < 0.001), 20 µM L-carnitine (p = 0.003) and
10 µM piracetam (p = 0.001) groups. A trend toward lower
percentage was also observed in the 20 µM compared with
the 10 µM L-carnitine group (p = 0.060) (Fig. 5).
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Protecting against sonoporation-induced bio-effects ● W.-H. Liao et al. 5

Fig. 2. Effect of ultrasound (US) intensity on L-carnitine- or piracetam-mediated transfection. NIH3T3 cells and BNL cells
were pre-treated with 20 µM L-carnitine or piracetam and then transfected with various intensities of US (cavitation index [CI] = 10
or 15). After 18 h of incubation, luciferase activity in NIH3T3 cells (a) and BNL cells (b) was analyzed. Luciferase activity is
represented as relative luminescence units (RLU) per milligram of protein. Relative cell viability in both NIH3T3 cells (c) and
BNL cells (d) was not reduced under sonoporation if cells were pre-treated with 20 µM L-carnitine or piracetam. *p < 0.05.

L-carnitine and piracetam prevent US-induced caspase-
3/7 activity in NIH3T3 cells
On mitochondrial membrane damage, mitochon-
drial cytochrome c is released into the cytoplasm.
Cytochrome c binds with APAF1 to form an apoptosome,
which mediates the activation of caspase-9, triggering a
cascade of caspase activation (such as caspase-3/7). As il-
lustrated in Figure 6, caspase-3/7 activity increased
significantly on US exposure, whereas pre-treatment
with L-carnitine and piracetam reversed this increase in
caspase-3/7 activity. These findings indicate that
sonoporation-induced caspase activity could be inhibited
by pre-treatment with L-carnitine and piracetam.
L-carnitine and piracetam ameliorate mitochondrial
ATP production rates after sonoporation
To investigate mitochondrial function after
sonoporation, we analyzed the ATP level. As illustrated
in Figure 7, the ATP level was significantly increased in
all non-US-exposed groups (all p values < 0.05). After
sonoporation, the ATP level was higher in cells pre-
treated with 20 µM L-carnitine or 20 µM piracetam than
in those not pre-treated (p = 0.019 and 0.011, respective-
ly). These results indicate that L-carnitine and piracetam
may promote ATP production to protect cells against US-
mediated stress.
DISCUSSION AND CONCLUSIONS
After US exposure, the amount of ROS increases sig-
nificantly within the cytosol, activating ryanodine receptors
(RyRs) on the endoplasmic reticulum (ER); these acti-
vated RyRs subsequently promote Ca2+ release from the
ER into the cytoplasm (Gorlach et al. 2015; Honda et al.
2004; Zhong et al. 2011). In addition, extracellular calcium
influx is directly induced by sonoporation at the same time
(Honda et al. 2004; Lentacker et al. 2014). Excess ROS
and Ca2+ attack the mitochondrial membrane, resulting in
the loss of Δψm and initializing the apoptotic pathway finally
leading to programmed cell death (Gorlach et al. 2015;
Honda et al. 2004; Pinton et al. 2008). Therefore, treat-
ment with antioxidants (such as N-acetyl-L-cysteine,
catalase and Pronalen-sensitive skin) (Basta et al. 2003;
Honda et al. 2004; Tomankova et al. 2014) or Ca2+ ch-
elators (BAPTA-AM [Abcam] and ethylene glycol-bis(β-
aminoethyl ether)-N,N,N’,N’-tetraacetic acid [EGTA])
(Honda et al. 2004; Hutcheson et al. 2010) can signifi-
cantly reduce US-induced cell death. Our results indicated
that US-mediated gene transfection was increased and
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6 Ultrasound in Medicine & Biology Volume ■■, Number ■■, 2018

Fig. 3. Effect of L-carnitine or piracetam on sonoporation-mediated apoptosis. NIH3T3 cells were pre-treated with two con-
centrations of L-carnitine or piracetam and then transfected with US (1 MHz, cavitation index = 15, 110 s). After 18 h of incubation,
apoptotic cells were detected with Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) and measured by flow
cytometry. The quantitative result (% apoptotic cells) for apoptosis was compiled based on the information on early apoptotic
cells (Annexin V-FITC positive/PI negative). *p < 0.05.

sonoporation-associated cell death was ameliorated by L-carnitine or piracetam. Pre-treatment with L-carnitine
L-carnitine or piracetam. Further findings indicated that or piracetam alleviated the negative effects of sonoporation,
sonoporation-induced intracellular ROS generation, loss such as excessive ROS generation and apoptosis, thus en-
of Δψm and caspase activity were significantly reduced by hancing US-mediated gene transfection. In addition, we
 ARTICLE IN PRESS
Protecting against sonoporation-induced bio-effects ● W.-H. Liao et al.
Fig. 4. Effect of L-carnitine or piracetam on intracellular level of
reactive oxygen species (ROS) after sonoporation. NIH3T3 cells
were pretreated with two concentrations of L-carnitine or piracetam
and then transfected with ultrasound (US, 1 MHz, cavitation
index = 15, 110 s). After 2 h of incubation, intracellular ROS were
detected by 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA)
and analyzed by flow cytometry. In all L-carnitine- or piracetam-
treated groups, there was no significant difference between cells
treated with US and those not treated. *p < 0.05.
used the enhanced green fluorescent protein (EGFP) re-
porter system to analyze the efficiency of gene transfection.
However, the percentages of EGFP-positive cells in US-
mediated gene transfections did not significantly differ
among all groups (data not shown). These results may in-
dicate that pre-treatment with either L-carnitine or piracetam
did not increase the efficiency of US-mediated gene trans-
fection, but can protect the cells from the negative effects
of sonoporation, thus increasing overall gene expression.
L-Carnitine may protect cells from the negative
impacts of sonoporation through the regulation of intra-
cellular Ca2+. Elevation of intracellular Ca2+ results from
sonoporation-associated influx (Hutcheson et al. 2010;
Fig. 5. Effect of L-carnitine or piracetam on mitochondrial mem-
brane potential (Δψm) after sonoporation. NIH3T3 cells were
pretreated with two concentrations of L-carnitine or piracetam and
then transfected with ultrasound (US, 1 MHz, cavitation index = 15,
110 s). After 2 h of incubation, Δψm was detected by JC-1 and ana-
lyzed by flow cytometry. *p < 0.05.
7
Fig. 6. Effect of L-carnitine or piracetam on sonoporation-
mediated caspase activity. NIH3T3 cells were pretreated with two
concentrations of L-carnitine or piracetam and then transfected with
ultrasound (US). After 18 h of incubation, caspase-3/7 activity was
analyzed. Caspase activity is represented as relative luminescence
units (RLU) per milligram of protein. *p < 0.05 compared with no
pre-treatment.
Lentacker et al. 2014) and ROS-induced release from the
ER (Pinton et al. 2008), and it is recognized as an impor-
tant messenger within the cytosol to regulate survival
(Pinton et al. 2008). A high level of intracellular Ca2+ will
induce cell apoptosis, and sonoporated cells can be rescued
from apoptosis through the addition of a Ca2+ chelator
(Hutcheson et al. 2010). L-Carnitine has been reported as
a calcium chelator within the cytoplasm and thus could
regulate biological function (Banihani et al. 2015).
Fig. 7. Effect of L-carnitine or piracetam on sonoporation-
mediated ATP production. NIH3T3 cells were pretreated with two
concentrations of L-carnitine or piracetam and then transfected with
ultrasound (US). After 2 h of incubation, the ATP level was deter-
mined. The ATP level is represented as relative luminescence units
(RLU) per milligram of protein. *p < 0.05 compared with US ex-
posure but no pre-treatment.
 ARTICLE IN PRESS
8 Ultrasound in Medicine & Biology
Excessive intracellular Ca2+, which may lead to pro-
grammed cell death, may be reduced by L-carnitine
chelation. In this study, L-carnitine could promote cell vi-
ability and downregulate caspase activity after sonoporation.
Previous studies reported that L-carnitine alleviated oxi-
dative damage through its antioxidant function (Arockia
Rani and Panneerselvam 2001) or its ability to activate en-
dogenous antioxidant components (Ye et al. 2010). Taking
these findings together, L-carnitine can reduce sonoporation-
associated excessive ROS generation and intracellular Ca2+
elevation and, finally, promote cell viability.
Piracetam may reduce sonoporation-induced cell death
through its antioxidant function. Although several studies
have indicated that piracetam does not possess radical-
scavenging properties (Bentue-Ferrer et al. 1989; Keil
et al. 2006) or needs to be administered at a high con-
centration (500–5000 µM) to exhibit slight scavenging
activity (Horvath et al. 2002), piracetam notably de-
creased the rate of mitochondrial superoxide generation
induced by simvastatin (Costa et al. 2013) and protected
against oxidative stress induced by additional H2O2, sodium
nitroprusside (SNP) or β-amyloid peptide (Aβ) (Keil
et al. 2006; Kurz et al. 2010; Leuner et al. 2010). In the
present study, piracetam was found to reduce US-
induced intracellular ROS generation. In addition, piracetam
decreased the activities of anti-oxidative enzymes in aged
mice, such as superoxide dismutase, glutathione peroxi-
dase and glutathione reductase (Keil et al. 2006). A
reduction in the demand for these anti-oxidative enzymes
may indicate a decrease in oxidative stress. These results
imply that piracetam may protect cells from apoptosis
by reducing oxidative stress, although further studies
should address the effect of piracetam on free radical
scavenging activity.
Previous studies have found that piracetam inter-
acts with the plasma membrane to enhance membrane
fluidity, which is reduced in the brain on aging or in Al-
zheimer’s disease (Eckert et al. 1999; Muller et al. 1997).
In addition, treatment with β-sitosterol may increase the
fluidity of the inner mitochondrial membrane and conse-
quently increase Δψm and mitochondrial ATP content, which
is associated with positive effects on mitochondrial func-
tion (Shi et al. 2013). When membrane fluidity was
increased by treatment with lidocaine and heat (42–
44 °C), US-mediated gene transfection efficiency was
significantly enhanced (Nozaki et al. 2003). It is be-
lieved that the enhancement of membrane fluidity makes
the plasma membrane more easily permeable by
sonoporation and that it also decreases membrane tension
and facilitates membrane repair (Nozaki et al. 2003).
However, a prior study indicated that cholesterol allevi-
ates atractyloside-induced mitochondrial permeability by
decreasing membrane fluidity (Colell et al. 2003). In the
present study, 20 µM piracetam partially reversed the loss
Volume ■■, Number ■■, 2018
of Δψm in sonoporated cells (Fig. 5). We believe that a
higher concentration of piracetam may have better pro-
tective efficiency after sonoporation by altering membrane
fluidity and promoting cell membrane resealing after
sonoporation-induced damage. Whether this protective
mechanism results from increasing or decreasing mem-
brane fluidity remains to be elucidated.
Impaired mitochondrial function leads to a reduc-
tion in ATP level. Previous studies found that treatment
with piracetam after induction of oxidative stress by SNP
exposure enhances ATP levels (Keil et al. 2006) and that
L-carnitine is able to increase ATP levels in normal mouse
thymocytes (Huang et al. 2012). These were compatible
with our results. Further, a higher concentration (20 µM)
of L-carnitine or piracetam seemed to reverse the nega-
tive effect of sonoporation. There might be certain
associations between increased ATP level and reduced
sonoporation-related cell damage, but caution should be
used in concluding the existence of a cause-and-effect
relationship.
Acknowledgments—This work was supported by Grant MOST 103-
2314-B-002-011-MY3 from the Ministry of Science and Technology
(MOST), Grant BN-105-PP-03 from the National Health Research In-
stitutes (NHRI) and Grant 106-N3660 from National Taiwan University
Hospital. We also thank the staff of the Biomedical Resource Core at
the First Core Laboratories, National Taiwan University College of Med-
icine, for technical assistance.
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