Journal of Drug Delivery Science and Technology 77 (2022) 103882

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Effect of ultrasonic parameters on gene transfection efficiency and cell

viability of the multifunctional microbubble in vitro
Tho Anh Thi Tran a, Toan Phi Nguyen a, Nhung Hong Thi Duong a, Duy Hieu Truong b,
Bac Xuan Nguyen a, Cuong Khac Bui c, Lap Thi Nguyen a, *
a
Hanoi University of Pharmacy, Hanoi, 10000, Viet Nam
b
Quang Binh Pharmaceutical Joint-stock Company, Quang Binh, 510000, Viet Nam
c
Vietnam Military Medical University, Hanoi, 10000, Viet Nam

A R T I C L E I N F O A B S T R A C T

Keywords: Studies have reported that microbubbles combined with focused ultrasound (FUS) can increase the membrane
Acoustic parameter permeability of tumor cells by triggering membrane perforation (sonoporation) to improve brain tumor-targeted
Brain tumor gene delivery. However, few studies focused on how to increase the efficiency of gene delivery to brain tumor
Focused ultrasound
cells with minor cell damage. The aim of this in vitro study was to find optimal parameters for efficient gene
Gene delivery
transfer into glioma cells using the VEGFR2-targeted cationic microbubbles gene vector (MB) for future brain
Microbubble
tumor-targeted gene delivery. Transfection efficiency and cell viability were used to determine the optimal
transfection parameters. Various MB concentrations (2 × 107 to 16 × 107/mL), post-transfection incubation
times (1–3 days), acoustic pressures (0–1400 kPa), duty cycles (0.5–5%), pulse repetition frequencies (PRF)
(0.5–2000 Hz) and cycle numbers (25–10000) were investigated. The results showed that the MB concentration
of 4 × 107 MB/ml and the incubation time of 2 days were optimal conditions for maximum gene transfection
efficiency and minimum cell damage. In addition, gene transfection efficiency and cell viability depended
strongly on the acoustic exposure conditions. The optimal FUS parameters include an acoustic pressure of 700
kPa, a duty cycle of 5%, a PRF of 5Hz, and a cycle number of 10000. These findings support those MB con­
centrations and FUS parameters of the MB-mediated gene delivery system influence gene transfer efficiency and
cell viability. They also describe our novel development of multifunctional MB for FUS-triggered gene delivery in
brain tumor cells.

1. Introduction
Brain cancer is one of the causes that make patients most likely die
from [1]. Conventional brain tumor treatments include surgery, radia­
tion, and chemotherapy. Adversities in brain tumor treatments are due
to numerous anatomical and physiological characteristics of the brain,
making it difficult for drugs to invade, such as endothelial cell imper­
meability, local enzymes, receptors, and, especially, the blood-brain
barrier [2]. It is urgent to develop a suitable drug delivery system to
effectively deliver anti-cancer drugs to tumor tissue to meet these
challenges.
Gene therapy is a promising approach to brain cancer treatment.
Successful gene therapy requires the safe and effective transport of
therapeutic genes across the cell membrane. Although viral vectors are
effective for gene transfection, the inflammatory and immunogenic
responses they have caused raise safety concerns, prompting the
development of non-viral delivery methods [3]. The development of
non-viral vectors, typically the gene-carrying microbubble system, has
improved gene transfer efficiency, specificity, and biosafety [4].
Microbubbles are gas-encapsulating particles that can be surrounded by
an electrically charged membrane capable of trapping nucleic acid
molecules.
Gene delivery by microbubbles, on the other hand, still has relatively
low efficiency compared to viral or other delivery methods such as lip­
ofection and electroporation [5]. This phenomenon likely results from
insufficient numbers of microbubbles within the focused ultrasound
(FUS) field or in direct contact with the cell membrane. Also, glioma
cancer cells are typically surrounded by normal neural cells, so a tumor
cell-targeted gene delivery platform needs to be developed to scale back
off-target effects. A previous study indicated that the structure of outer

* Corresponding author.
E-mail address: lapnt@hup.edu.vn (L.T. Nguyen).

https://doi.org/10.1016/j.jddst.2022.103882
Received 19 June 2022; Received in revised form 2 October 2022; Accepted 7 October 2022
Available online 13 October 2022
1773-2247/© 2022 Elsevier B.V. All rights reserved.
T.A.T. Tran et al.
microbubbles’ shells allows them to construct covalent bonds with
various ligands, which establishs a novel targeted microbubble gene
delivery system [6]. Therefore, a more refined design that involves
modifying particular antibodies or ligands that bind to
disease-associated molecular markers expressed on vascular endothelial
cells of the brain tumor is essential to promote local aggregations of
microbubbles to increase gene transfection efficiency.
The vascular endothelial growth factor receptor 2 (VEGFR2) among
angiogenesis molecular markers is a key player. VEGFR2 is a high-
affinity receptor for vascular endothelial growth factor-A (VEGF-A)
[7]. VEGFR2 expression has been associated with tumor growth and
aggressiveness in various cancers [8]. VEGFR2 is also highly expressed
in tumor cells and endothelial cells in blood vessels that nurture brain
tumors [9]. Thus, attaching exogenous antibodies against VEGFR2 to the
microbubble’s outer shell is a promising strategy to target specific sites
in the vicinity of tumors to unload therapeutic genes.
In addition, the combination of microbubbles and FUS increases gene
transfer efficiency. The principle is that microbubbles are vehicles to
deliver therapeutic genes, and ultrasound forms “openings” between
cells by ultrasonic-activated oscillations, which also break the micro­
bubbles [10]. The cavitation effects from circulating microbubbles can
produce local intracellular gap junctions of the cerebral vesicular
endothelial cells, endocytotic cellular uptake, and vessel disruption.
Through sonoporation, microbubble cavitation may enhance and
develop drug delivery routes, allowing pharmaceutical substances to be
taken up. In particular, microbubbles oscillate steadily or violently
grow/collapse in response to ultrasound depending on the applied
acoustic pressure. Dynamic microbubble motion combined with fluid
motion can exert mechanical stresses on the surrounding tissues,
resulting in reversible cell membrane penetration that can help with
medication administration. Additionally, intense microbubble collapse
(inertial cavitation) causes secondary effects such as micro-jet and
shockwaves, which enhance delivery capacity. Cavitation-induced ef­
fects can result in cell morphology changes and long-term bio-effects (e.
g., temporal cell permeability changes, cell lysis), which can help
improve therapeutic agent delivery and uptake [11].
Moreover, the results obtained from a previous study indicated that
each tumor cell line has its optimum parameter combination and opti­
mum sonoporation efficiency [12]. Another study also revealed that
transfection efficiency and loss of cell viability could be attributed to the
chosen ultrasound parameters [13]. Therefore, proper optimization of
ultrasound parameters must be done to achieve therapeutic results and
minimize unwanted damage to healthy tissues.
In a previous study, a multifunctional microbubble system that
carries both a gene encoding luciferase and an anti-VEGFR2 antibody to
target brain tumors was fabricated [14]. However, the quantitative
dependence of sonoporation on FUS exposure parameters was not well
documented and, therefore, is the subject of this study. The gene
encoding luciferase, an enzyme that produces bioluminescence, has
been used for detection and biological effect evaluation. In combination
with FUS, the influence of microbubbles’ concentrations and acoustic
exposure parameters on gene transfection and cell viability using the
microbubble system mentioned above was determined in vitro.
2. Materials and methods
2.1. Materials
The lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyeth­
ylene glycol)-2000] (DSPE-PEG 2000), 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE–
PEG2000–Biotin) and 1,2-dipalmitoyl-3-trimethylammonium-propane
(DPTAP), were obtained from Avanti Polar Lipids, AL, USA. Avidin
was purchased from Sigma Aldrich, MO, USA. CD309 (VEGFR-2) biotin,
a human monoclonal antibody (0.5 mg/ml), was obtained from Miltenyi
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Journal of Drug Delivery Science and Technology 77 (2022) 103882
Biotec (Germany). Alarma blue assay was purchased from AbDSerotec,
Oxford, UK. The red firefly luciferase gene (pLUC, 6.6 kb) was purchased
from Origene Technologies, USA. Plasmid Maxi Kit was obtained from
Macherey-Nagel, Germany. The U87 glioma cell line was obtained from
American Type Culture Collection. All other reagents were of analytical
grade.
2.2. Plasmid preparation
An expression vector for the pLUC was all driven by a cytomegalo­
virus (CMV) promoter. Plasmid DNA was purified from the culture of
Escherichia coli DH5a with the Plasmid Maxi Kit, following the protocol
provided by the manufacturer. The plasmid DNA purity was assessed by
measuring absorption at 260 nm wavelength (A260) by NanoDrop
(NanoDrop 2000, Thermo Fisher Scientific, IL, USA). The A260:A280
ratio of plasmid DNA was between 1.8 and 1.9. The quality of the pu­
rified plasmid was further analyzed by enzyme restriction and subse­
quent electrophoresis on a 0.8% agarose gel.
2.3. Preparation of DNA-loaded VEGFR2 targeted cationic microbubbles
The cationic microbubbles with anti-VEGFR2 antibodies were pre­
pared using the thin-film hydration technique described in a previous
study [14]. Firstly, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyeth­
ylene glycol)-2000] (DSPE-PEG 2000), 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE–
PEG2000–Biotin) and 1,2-dipalmitoyl-3-trimethylammonium-propane
(DPTAP) were dissolved in chloroform at a weight ratio of 9:1:1:1, and
drained via rotary evaporator to form a dried film. Secondly, the film was
hydrated with a 5 μl/ml glycerol-PBS buffer in an airtight vial. The gas in
the vial was removed and refilled with perfluoropropane (C3F8) gas.
Thirdly, the vial was shaken by an agitator for 45 s to produce micro­
bubbles with a hydrophobic gas core and lipid exterior. To eliminate free
lipids, biotinylated microbubbles were centrifuged for 2 min at 6000 rpm
(Mini-micro centrifuge, Bertec Enterprise Co., Ltd., Taiwan) and resus­
pended in glycerol-PBS buffer.
Fourthly, the biotinylated microbubbles were then incubated for 10
min at room temperature with 1 mg of avidin (in an aqueous solution of
10 mg/ml). Next, the microbubbles were centrifuged again to eliminate
excess avidin and resuspended in glycerol-PBS buffer. Fifthly, 75 μg of
CD309 (VEGFR-2) biotinylated antibody was added to the avidin-
biotinylated microbubble suspension and incubated for 10 min. The
microbubbles were centrifuged again and resuspended to remove excess
biotinylated antibodies. Finally, an appropriate amount of pLUC in
deionized water was incubated with the microbubbles by slowly mixing
for 30 min to speed up DNA adsorption onto the microbubble surface.
Hence, DNA-loaded VEGFR2 targeted cationic microbubbles (MB) were
formed.
2.4. Evaluating pLUC gene transfection and cell viability in vitro
Gene transfection and cell viability were assessed using U87 glioma
cells with a modified protocol based on previous studies [15,16]. More
specifically, U87 glioma cells were cultured in Dulbecco’s Modified
Eagle’s medium/Ham’s Nutrient Mixture F-12 (DMEM/F12), supple­
mented with 10% fetal bovine serum, penicillin/streptomycin at 37 ◦ C.
The day before the experiments, 4 × 104 cells per well were seeded in a
24-well plate. Then, the VEGFR2-targeted cationic microbubbles were
incubated with 2 μg pLUC in 4 ml DMEM for 30 min at room tempera­
ture. After the medium in a 24-well plate was removed, DNA-loaded MB
were added to each well. An inverted setup promoted cell targeting ef­
ficiency by providing close contact between floated MB and cells for 10
min and then turning upright for FUS treatment. Next, serial acoustic
parameters of FUS (acoustic pressure, cycle number, and pulse repeti­
tion frequencies (PRF)) and concentration of MB (2-16 × 107 MB/mL)
T.A.T. Tran et al.
were applied to optimize the transfection efficiency. FUS exposure
(acoustic pressures of 250–1400 kPa, cycle numbers of 25–10000, PRF
of 0.5–2000 Hz, duty cycles of 0.0125–5%, and exposure duration of 1
min) was applied with a 6 mm diameter FUS probe (model V302,
Olympus NDT Inc., MA, USA). An RF power amplifier (150A100B, AR,
PA, USA) was used to generate the sonication. Four hours following FUS
sonication, the cells were washed with fresh PBS, and a fresh culture
medium was then added. A minimum of 15 replicate data points were
collected at each tested condition. The pLUC expression and cell
viability were individually measured by bioluminescence imaging and
Alarma blue assay.
For bioluminescence assays, pLUC transfected cells were measured
1–3 days following ultrasound exposure by bioluminescence imaging
(IVIS-200, Xenogen Corporation, Ca, USA). Thirty seconds before im­
aging, D-luciferin (150 μg/mL) was added to each well (Biosynth AG,
Staad, Switzerland). Photon flux for each well was measured at 1.5 min
after adding D-luciferin by an IVIS charge-coupled device camera. Serial
1-min image frames were collected until the bioluminescent signal peak
was reached. Then, image analysis of all frames was performed by
quantifying the average radiance (photon/s/cm2/steradian, p/s/cm2/
sr) of each wall via Living Image 3.0 software (Caliper Life Sciences, MA,
USA).
The cell viability was converted by the percentage of formazan
production assaying the Alarma blue assay with a plate reader (Tecan
Infinite M200, Tecan Trading AG, Switzerland) at an absorbance
wavelength of 600 nm. Alarma blue (5 mg/mL in serum-free medium)
was added to each well and incubated at 37 ◦ C for 2 h. The absorbance of
each plate was measured with a fluorescence plate reader system at a
wavelength of 570 nm and a reference wavelength of 600 nm. The cell
viability was calculated according to the formula provided below.
(εOX )600nm × A570nm − (εOX )570nm × A600nm
cell viability (%) =
(εOX )600nm × A0,570nm − (εOX )570nm × A0,600nm
Where εox = molar extinction coefficient of Alamar Blue™ oxidized form
(blue), A = absorbance of experimental wells, and A0 = absorbance of
positive growth control wells (cells with Alamar Blue without test
agent).
2.5. Statistical analysis
Data are presented for each condition as mean ± standard deviation
(SD) from three separate experiments. All statistical tests were per­
formed using R Statistical Software (version 2.14.0). Statistical signifi­
cance of observed effects was determined by analysis of variance
(ANOVA) and Post hoc Tukey tests. A p-value < 0.05 was considered
statistically significant.
Fig. 1. (A) Gene transfection efficiency and (B) cell viability under various microbubble concentrations and post-transfection incubation time. The two-
way ANOVA test showed that each group of microbubble concentration and time after ultrasound exposure demonstrated luciferase expression statistically sig­
nificant (ANOVA, p < 0.001) and cell viability statistically significant (ANOVA, p < 0.001).
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Journal of Drug Delivery Science and Technology 77 (2022) 103882
3. Results
3.1. Effect of MB concentration and post-transfection incubation time
The cells were exposed to FUS at a center frequency of 1 MHz and a
constant acoustic pressure of 700 kPa, a cycle number of 10000, a PRF of
5Hz, a duty cycle of 5%, exposure time of 1 min, and 1.5 μg pLUC per
well. A range of MB concentrations ((2-16) × 107 MB/ml) and post-
transfection incubation times (1, 2, and 3 days) were evaluated for
their effects on gene transfection efficiency and cell viability. As shown
in Fig. 1A, the transfection efficiency increased first and then decreased
with the increase of microbubble concentrations. On day one and day
two post-transfection, the luciferase expression reached a peak at an MB
concentration of 4 × 107 MB/ml. However, after three days post-
transfection, the luciferase expression reached a peak at an MB con­
centration of 8 × 107 MB/ml. On the other hand, the results from Fig. 1B
showed that the cell viability tended to decrease with increasing con­
centration of microbubbles: the highest was at the MB concentration of
2 × 107 MB/ml, then gradually decreased to the lowest at 16 × 107 MB/
ml (Tukey HSD, p < 0.001).
The highest average radiances on day one and day two post-
transfection were (14.46 ± 2.35) × 103 and (18.52 ± 1.55) × 103 (p/
s/cm2/sr), respectively. At the MB concentration of 4 × 107 MB/ml, the
cell viability remained above 70%. However, the highest average radi­
ance on day three post-transfection was (18.04 ± 0.39) × 103 (p/s/cm2/
sr) at the MB concentration of 8 × 107 MB/ml, while the cell viability
was quite low (45.77 ± 0.93%). Considering the transfection efficiency
and cell viability, the MB concentration of 4 × 107 MB/ml and the time
of two days post-transfection were determined as optimal for subsequent
experiments.
3.2. Effect of acoustic pressure
The inertial cavitation dose (ICD) and subharmonic intensity were
measured in this experiment. Investigating the acoustic properties of
MB, the thresholds for both stable and inertial cavitation of MB occurred
at 250 kPa and 350 kPa, respectively (Fig. 2).
The influence of acoustic pressure on gene transfection efficiency
and cell viability was further investigated with varied acoustic pressures
(250–1400 kPa). Cells were insonated with FUS at a center frequency of
1 MHz and a constant cycle number of 10000, PRF of 5 Hz, the duty
cycle of 5%, exposure time of 1 min, the MB concentration of 4 × 107
MB/ml, and 1.5 μg of pLUC per well. The luciferase expression of cells
was observed on day two post-transfection. The results are presented in
Fig. 3.
As shown in Fig. 3A, increasing acoustic pressure from 250 to 400
kPa provided no significant increase in luciferase expression (group a,
Tukey HSD, p > 0.05). After that, increasing the acoustic pressure from
T.A.T. Tran et al. Journal of Drug Delivery Science and Technology 77 (2022) 103882

acoustic pressure from 700 kPa to 900 kPa (group b and c, Tukey HSD, p
< 0.001). Taken together, Fig. 3A and B suggested that we should keep
the optimal acoustic pressure at 700 kPa for all subsequent tests.

3.3. Effect of PRF, duty cycle, and cycle number

3.3.1. Effect of PRF and duty cycle

Fig. 2. Inertial cavitation dose and subharmonic intensity of gene-loaded
microbubbles under various acoustic pressure.
400 kPa to 700 kPa resulted in a significant increase in luciferase
expression (group a, b, and c, Tukey HSD, p < 0.001). However,
increasing the acoustic pressure from 700 kPa to 1400 kPa resulted in a
significant decrease in luciferase expression (group c, d, e, and f, Tukey
HSD, p < 0.001).
Meanwhile, in Fig. 3B, the cell viability showed a significant
decrease with the increase in acoustic pressure. When the acoustic
pressure increased from 300 kPa to 700 kPa, the cell viability decreased
but remained above 70% (group a and b, Tukey HSD, p < 0.001).
However, there is a sudden decrease in cell viability when increasing the
The PRF is an important parameter of burst waveforms. The effect of
PRF (0.5–5Hz) and duty cycle (0.5–5%) on the gene transfection effi­
ciency and cell viability are shown in Fig. 4. Cells were insonated with
FUS at a center frequency of 1 MHz and a constant acoustic pressure of
700 kPa, cycle number of 10000, exposure time of 1 min, with the MB
concentration of 4 × 107 MB/ml, and 1.5 μg pLUC per well.
The result clarified that increasing the PRF and duty cycle while
maintaining a constant cycle number of 10000 resulted in a significant
increase in luciferase expression (groups a, b, and c, Tukey HSD, p <
0.001) (Fig. 4A) and a decrease in cell viability (group d, a, b, and c,
Tukey HSD, p < 0.001) (Fig. 4B). As shown in Fig. 4A and B, the
transfection efficiency increased with the increase of PRF and duty cycle
until reaching its highest level, as PRF and duty cycle were 5 Hz and 5%,
respectively, while the cell viability remained close to 70%.
3.3.2. Effect of PRF and cycle number
Cells were insonated with FUS at the center frequency of 1 MHz and
constant acoustic pressure of 700 kPa, the duty cycle of 5%, exposure
time of 1 min, the MB concentration of 4 × 107 MB/ml, and 1.5 μg pLUC
per well. The effect of PRF and cycle number is shown in Fig. 5.
Increasing the cycle number from 25 to 10000 while reducing the
PRF from 2000 Hz to 5 Hz resulted in a significant increase in luciferase

Fig. 3. (A) Gene transfection efficiency and (B) cell viability under various acoustic pressure. A. Gene transfection efficiency (a, b, c, d, e, f: different subgroups
that are statistically significant (Tukey HSD, p < 0.001)); B. Cell viability (d, a, b, c: different subgroups that are statistically significant (Tukey HSD, p < 0.001)).

Fig. 4. (A) Gene transfection efficiency and (B) cell viability under various duty cycles and pulse repetition frequencies (PRF). A. Gene transfection effi­
ciency (a, b, c: different subgroups that are statistically significant (Tukey HSD, p < 0.001)); B. cell viability (d, a, b, c: different subgroups that are statistically
significant (Tukey HSD, p < 0.001)).

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T.A.T. Tran et al. Journal of Drug Delivery Science and Technology 77 (2022) 103882

Fig. 5. (A) Gene transfection efficiency and (B) cell viability under various pulse repetition frequencies (PRF) and cycle numbers. A. Gene transfection
efficiency (a, b, c, d: different subgroups that are statistically significant (Tukey HSD, p < 0.001)); B. cell viability (a, b, c: different subgroups that are statistically
significant (Tukey HSD, p < 0.001)).

expression, as shown in Fig. 5A (group a, b, c, and d, Tukey HSD, p <
0.001). On the other hand, this caused a significant decrease in cell
viability (group a, b, and c, Tukey HSD, p < 0.001) (Fig. 5B). The gene
transfection efficiency reached a maximum level when the PRF and
cycle numbers were 5 Hz and 10000, respectively, while the cell
viability remained close to 70%.
3.3.3. Effect of cycle number and duty cycle
Cells were insonated with FUS at the center frequency of 1 MHz and
constant acoustic pressure of 700 kPa, PRF of 5 Hz, exposure time of 1
min, and the MB concentration of 4 × 107 MB/ml and 1.5 μg pLUC per
well. The effect of cycle number and duty cycle is shown in Fig. 6.
The results showed that increasing the cycle number and duty cycle
resulted in a significant increase in luciferase expression (group a, b, c,
and d, Tukey HSD, p < 0.001 (Fig. 6A) and a decrease in cell viability
(group c, a, and b, Tukey HSD, p < 0.001) (Fig. 6B). In addition, the
highest transfection efficiency achieved at cycle number and duty cycle
were 10000 and 5%, respectively, while the cell viability remained close
to 70%.
Therefore, with the results from Figs. 4–6, we selected a cycle
number of 10000, a duty cycle of 5%, and a PRF of 5Hz as the optimum
parameters for the follow-up experiments.
number of 10000, a PRF of 5 Hz, a duty cycle of 5%, exposure time of 1
min, the MB concentration of 4 × 107 MB/ml, 1.5 μg pLUC per well, and
a luciferin concentration of 150 μg/ml. We examined the groups that
lacked one or more than one of the treatments, including FUS, MB,
pLUC, and luciferin.
Luciferin, the luciferase substrate, was responsible for the charac­
teristic light emission. The luciferase expression and cell viability are
shown in Fig. 7. There was no luciferase expression in groups where cells
lacked one or more treatments, including FUS, MB, pLUC, and luciferin.
Group 7 showed that no gene delivery was observed without MB, indi­
cating that FUS alone does not affect gene delivery. In addition, in the
absence of FUS (group 8), no gene delivery occurred under any condi­
tions, confirming that MB alone cannot deliver the gene. Group 8 still
showed no luciferase expression because MB size (≈1 μm) was too large
and too solid for cell endocytosis and gene transfection. This indicated
that cavitation and sonoporation triggered by FUS were required for
gene transfection in cells. When these components were sufficient, the
gene transfection efficiency increased sharply in group 9, demonstrating
that these factors were unlikely to be independent, and the process was
strongly related to cavitation and cavitation-induced activities. Cell
viability varied between groups but remained around 70%. Group 5 and
9 showed lower cell viability due to MB cavitation.

3.4. Effect of other exposure factors 4. Discussion

After choosing optimal data from the six parameters discussed above Microbubble-mediated ultrasound has great potential for clinical
(MB concentration, time after ultrasound exposure, acoustic pressure, translation in oncology because it is safe and non-invasive. This
PRF, duty cycle, and cycle number), cells were insonated with FUS at a approach can create temporary and reversible openings in vessel walls
center frequency of 1 MHz and a constant pressure of 700 kPa, a cycle and cellular membranes through sonoporation, allowing enhanced

Fig. 6. (A) Gene transfection efficiency and (B) cell viability under various cycle numbers and duty cycles. A. Gene transfection efficiency (a, b, c, d: different
subgroups that are statistically significant (Tukey HSD, p < 0.001)); B. Cell viability (c, a, b: different subgroups that are statistically significant (Tukey HSD, p
< 0.001)).

5
T.A.T. Tran et al.
Fig. 7. Gene transfection efficiency and cell viability under exposure factors.
**: cell viability and gene transfection efficiency are statistically significant (Tukey HSD, p<0.01).
transport of therapeutic agents across these biological barriers in the
insonated region. Most studies have shown successful delivery of
ultrasound-guided microbubble systems. However, the reported de­
livery efficiency is inconsistent. Thus, parameter optimization may need
to be assessed to enable maximum gene transfection efficiency and
minimum cell death. This study tested the hypothesis that varying
acoustic parameters of FUS-mediated gene delivery by MB influence
efficiency and cell viability to find the most suitable parameters for
efficient gene transfection on glioma cells for future brain tumor-
targeted gene therapy. The effects of microbubble concentration, time
after ultrasound exposure, and FUS parameters (acoustic pressure, duty
cycle, PRF, and cycle number) on gene transfection efficiency and cell
viability were investigated in a glioma cell line. We chose the U87 gli­
oma cell line because human glioblastomas are thought to be initiated
by glioma stem-like cells, and these cells express higher levels of VEGF
receptor 2 (VEGFR-2) [17]. Therefore, this VEGFR2-targeted MB was
fabricated to increase gene delivery efficiency by taking advantage of
the active targeting capability.
Prior to evaluation of cell viability and gene transfection, physical
characteristics of the tested microbubble have been assessed, which
include the binding efficiency of antibodies onto the microbubble’s
surface (data not shown). The amount of antibody added to create the
targeted microbubbles was excessive enough compared to the total
estimated area of microbubbles. Thus, we indirectly assumed that the
number of antibodies on the surface of each microbubble was abundant
enough to target the cells.
The experimental conditions used appear critical for efficient gene
expression in cells. First, we looked at the effect of MB concentration on
gene delivery efficiency. Fig. 1 indicated that the transfection capacity
tended to gradually increase to a threshold and then decrease over time
at all concentrations. Meanwhile, cell viability tended to decrease over
time when increased microbubble concentration. This result can be
explained by lower cell viability, so these damaged cells were unable to
increase gene expression. Too high a concentration of microbubbles can
cause cytotoxicity and mass cell death and reduce the expression of the
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Journal of Drug Delivery Science and Technology 77 (2022) 103882
luciferase gene. This trend is similar to the results shown in some pre­
vious studies [12,18–22]. For instance, Shapiro et al. found that the
concentration of microbubbles must be kept in a very tight range to
achieve enhanced sonoporation. Their results indicated that delivery of
5 × 105 MB/ml, which is lower than our result of 4 × 107 MB/ml, led to
significantly enhanced transfection compared to lower and higher dos­
ages of microbubbles. Below this concentration, there may be too little
impact from microbubble oscillation, and the microbubbles may be
more rapidly destroyed [18]. However, if the microbubble concentra­
tion is too high, the microbubbles may shield each other from the
acoustic field, and the effect would be attenuated. Also, too many
microbubbles could cause increased tissue damage [18].
Results from Fig. 1 also initially evaluated the effect of time after
gene transfer on the efficiency of MB combined with FUS. The cell
viability tended to decrease with post-transfection incubation time with
increasing microbubble concentration, probably due to increased
membrane damage with larger microbubble-to-cell ratios. However,
within the limitations of the study, it cannot be conducted at the next
time point after gene transfection (after three days), so the study has not
proven the specific mechanism of changes in the gene-delivery system
over time, as well as where is the most appropriate timeline for gene
transfection and cell viability. In the subsequent study, we will conduct a
more detailed analysis of the effect of time (at more time points) to find
out the mechanism of the microbubble system combined with
ultrasound.
Next, we examined the acoustic properties of MB. Stable cavitation
and inertial cavitation of MB started at 250 kPa and 350 kPa, respec­
tively (Fig. 2). Results also indicated that subharmonic intensity and ICD
were in a strong positive correlation with acoustic pressure. An increase
in acoustic pressure under the threshold of inertial cavitation caused
non-linear expansion and compression of the MB and resulted in the
emission of non-linear harmonic signals at multiples of the transmitted
frequency, which was known as stable cavitation. Also, the oscillating
MB generated steady microstreaming and large shear forces [23].
Higher acoustic pressure over the threshold caused forced expansion
T.A.T. Tran et al.
and compression of MB, resulting in bubble disruption.
Furthermore, increasing the acoustic pressure from 400 kPa to 700
kPa resulted in a significant increase in luciferase expression (groups b
and c, Tukey HSD, p < 0.001). This result indicated that an acoustic
pressure threshold greater than 400 kPa was required for gene trans­
fection because inertial cavitation of MB started from 400 kPa (Fig. 3A).
However, increasing the acoustic pressure from 700 kPa to 1400 kPa
resulted in a noticeable decrease in luciferase expression (groups d, e,
and f, Tukey HSD, p < 0.001). Such a reduction in gene transfection is
that the cells have reached the cytotoxic threshold (Fig. 3B). Increasing
sonoporation also resulted in a larger proportion of cells that are unable
to repair their damaged cell membranes [12,24,25]. The reason may be
that high acoustic pressure will enhance the cytotoxic process, as pre­
viously proven in some studies [26–31]. Cell membrane permeability
increases with decreasing acoustic pressure, and viability falls. The
number of permeabilized cells, on the other hand, decreases as the
pressure amplitude increases. Cells do not appear to be permeabilized or
destroyed below a certain level. Higher than a threshold of 2–3 MPa
acoustic pressures (higher than our result) may be less favorable for
producing reversible permeabilization, according to R. Karshafian et al.
[27]. Therefore, gene expression and cell viability should both be
evaluated when optimizing acoustic pressure. To sum up, an acoustic
pressure threshold of 400 kPa is required for the microbubble transport
system to start gene-transferring and gene transfection efficiency rea­
ches its peak at a negative pressure of 700 kPa.
Furthermore, a similar increasing trend as acoustic pressure is also
observed with PRF, duty cycle, and cycle number increasing (Figs. 4–6).
When investigating the effects of the duty cycle (a gradual increase from
0.5% to 5%) and the PRF (a gradual increase from 0.5 Hz to 5 Hz) on
luciferase gene expression and cell viability, we found that increasing
the duty cycle from 0.5% to 5% and the PRF from 0.5 Hz to 5 Hz showed
the average bioluminescence level tended to increase rapidly. Mean­
while, the ability to transfer genes tends to decrease. At a duty cycle
value of 5% and a PRF of 5 Hz, the mean bioluminescence was highest,
and cell viability was lowest. However, at these values, the cell viability
was still higher than 70% (Fig. 4). The higher the duty cycle was, the
greater inertial cavitation was created, where a higher percentage of
microbubbles lasted longer between pulses.
Similarly, in Fig. 5, the higher cycle number generated greater in­
ertial cavitation, where a higher level of microbubbles endured longer
between pulses. This result can be explained by the increased number of
focused ultrasound cycles, leading to the formation of a larger mem­
brane hole, creating an effect that helps the microbubble to be more
stable in the interval between pulses. This is an essential factor deter­
mining the success of using multifunctional transport systems in future
in vivo model studies. The importance of cycle numbers might be a
valuable characteristic of microbubbles. More control over the degree of
sonoporation in vivo may be achieved by increasing cycle number to
improve sonoporation or reducing cycle number to ameliorate cell death
in more sensitive tissues.
Results from Fig. 6 demonstrated that luciferase expression was
heavily dependent on cycle number, with more transfection efficiency at
a higher cycle number of 10000. It can be concluded that the influence of
ultrasound parameters such as cycle numbers, PRF, and duty cycles on
the gene transfection of microbubbles carrying genes encoding lucif­
erase in vitro.
In general, acoustic pressure, cycle number, duty cycles, and PRF are
directly proportional to gene transfection and inversely proportional to
cell viability (Figs. 3–6), which is also the case for previous conclusions
[25,32]. The overall trend for the data is that increases in transfection
efficiency are associated with decreased cell viability. These experi­
mental observations strongly suggest that optimization is needed since
the maximum transfection rate and cell viability cannot be obtained
together. The justification may be that these increased ultrasound pa­
rameters change membrane pore size, thereby facilitating gene transfer.
Nonetheless, increasing these parameters to too high may tend to
7
Journal of Drug Delivery Science and Technology 77 (2022) 103882
saturate or eventually decrease as extremely large membrane pores are
created, which is considered irreparable damage to most cell mem­
branes. These pores allow macromolecules to pass and subsequently
cause cell lysis [33]. Therefore, optimizing the parameters for
microbubble-mediated ultrasound transfection is inevitable to have a
safe and efficient transport of therapeutic materials.
There is a limitation associated with our study. The study was con­
ducted only on one glioma cell line, namely U87 glioma cell line. It
would be interesting to determine whether our results of optimal ul­
trasonic parameters are the case for other glioma cell lines or not. Hence,
further studies need to be done on additional human glioma cell lines.
However, we contend that a similar trend of cell viability and gene
transfection efficiency in other glioma cell lines would also be observed
due to sonoporation. In addition, the reason why we chose U87 cell line
is because it is the most widely used human glioblastoma cell line in
brain cancer research. Glioblastoma multiforme is also the most com­
mon malignant brain cancer. Therefore, in this study, we want to focus
on U87 cell line only.
5. Conclusion
This study assessed the effects of microbubble’s concentration, post-
transfection time, and ultrasound parameters (acoustic pressure, duty
cycle, PRF, cycle number) on gene transfection and cell viability. The
effect of each microbubble’s concentration and time after ultrasound
exposure on gene transfection and cell viability are all statistically sig­
nificant (two-way ANOVA, p < 0.001). In vitro models also show that the
higher the duty cycle, PRF, and cycle number are, the more efficiently
the gene transfection is conveyed (Tukey HSD, p < 0.001). Simulta­
neously, cell viability decreases gradually (Tukey HSD, p < 0.001).
Together, on day two post-transfection, microbubble concentration of 4
× 107 MB/mL, acoustic pressure of 700 kPa, the duty cycle of 5%, PRF of
5 Hz, and cycle number of 10000, the gene transfection and cell viability
of microbubbles transport system combined with ultrasound are the
most optimal in our experiment setting. This is the basis for conducting
further studies to evaluate the gene transfection of the microbubbles
transport system combined with ultrasound in targeting tumors in vivo.
Funding
This work was supported by the Ministry of Science and Technology
of Vietnam (Code: NDT.65.TW/19).
Author statement
Tho Anh Thi Tran: Conceptualization, Methodology, Investigation,
Formal analysis, Writing - Original Draft. Toan Phi Nguyen: Writing -
Original Draft, Writing - Review and Editing, Visualization. Nhung Hong
Thi Duong: Investigation, Data Curation, Visualization. Duy Hieu
Truong: Writing - Review and Editing. Bac Xuan Nguyen: Supervision.
Cuong Khac Bui: Resources. Lap Thi Nguyen: Funding acquisition,
Project administration.
Declaration of competing interest
None.
Data availability
The data that has been used is confidential.
Acknowledgments
The authors wish to convey their gratitude for the financial support
from the Ministry of Science and Technology of Vietnam and helpful
expert support from Prof. Chih-Kuang Yeh and his lab members (En-Ling
T.A.T. Tran et al.
Chang and Ching-Hsiang Fan), National Tsinghua University, Taiwan.
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