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Ultrasound-Enhanced siRNA Delivery Using Magnetic
Nanoparticle-Loaded Chitosan-Deoxycholic Acid
Nanodroplets
Jeong Yu Lee, Calum Crake, Boon Teo, Dario Carugo, Marie de Saint Victor, Anjali Seth,
and Eleanor Stride*
DOI: 10.1002/adhm.201601246
Dr. J. Y. Lee, Dr. C. Crake, Dr. B. Teo, Dr. D. Carugo,
Dr. M. de Saint Victor, Dr. A. Seth, Prof. E. Stride
Institute of Biomedical Engineering
Department of Engineering Science
University of Oxford
Oxford, Old Road, Campus OX3 7DQ, UK
E-mail: eleanor.stride@eng.ox.ac.uk
Dr. D. Carugo
Faculty of Engineering and the Environment
Southampton University
Southampton SO17 1BJ, UK
Small interfering RNA (siRNA) has significant therapeutic potential but its
clinical translation has been severely inhibited by a lack of effective delivery
strategies. Previous work has demonstrated that perfluorocarbon nanodro-
plets loaded with magnetic nanoparticles can facilitate the intracellular
delivery of a conventional chemotherapeutic drug. The aim of this study is to
determine whether a similar agent can provide a means of delivering siRNA,
enabling efficient transfection without degradation of the molecule. Chitosan-
deoxycholic acid nanoparticles containing perfluoropentane and iron oxide
(d0 = 7.5 ± 0.35 nm) with a mean hydrodynamic diameter of 257.6 ± 10.9 nm
are produced. siRNA (AllStars Hs cell death siRNA) is electrostatically bound
to the particle surface and delivery to lung cancer cells and breast cancer cells
is investigated with and without ultrasound exposure (500 kHz, 1 MPa peak-
to-peak focal pressure, 40 cycles per burst, 1 kHz pulse repetition frequency,
10 s duration). The results show that siRNA functionality is not impaired by
the treatment protocol and that the nanodroplets are able to successfully
promote siRNA uptake, leading to significant apoptosis (52.4%) 72 h after
ultrasound treatment.
increase in the efficacy of conventional
chemotherapeutic agents.[1] To date, how-
ever, the potential of siRNA has not been
realized clinically due to its inefficient sys-
temic delivery. Notably, in its naked form,
siRNA suffers from poor pharmacokinetics
due to its low bloodstream stability and
rapid glomerular filtration. Furthermore,
target cell transfection efficiency is very
low because it has no intrinsic mecha-
nism for cell entry. In vitro this limitation
can be overcome with the use of positively
charged polymer-based delivery vectors, but
in vivo these vectors are too unstable and
the electrostatic mediated cell entry mecha-
nism upon which they rely is too unspe-
cific.[2] While liposomal delivery systems
can provide enhanced circulation time thus
benefiting from the enhanced permeability
and retention effect, they achieve very lim-
ited penetration beyond the perivascular
space of the tumor and do not efficiently
release their cargo.[3]
There have been a number of recent studies demonstrating
the potential of ultrasound as a means of promoting siRNA
delivery both in vitro and in vivo.[4,5] Ultrasound mediated
delivery can be significantly enhanced through the use of
gas microbubbles that provide a means of encapsulating and
improving tissue penetration and uptake of various thera-
peutics, including siRNA. For example, Carson et al. used
microbubbles for inhibition of endothelial growth factor
(EGF) receptor signaling, an established strategy for treating
numerous types of cancer. Their results indicated that tumor
growth could be decelerated, arrested or even reversed in
EGFR-treated mice.[4] Florinas et al. similarly showed that
microbubbles loaded with PEI (polyethylamine) encapsulating
siRNA could induce knockdown of vascular EGF in vitro and
decelerate tumor growth in vivo in a mouse model.[6] A further
advantage of this approach is that the timescales for treatment
are significantly reduced compared to passive delivery methods.
Typically, ultrasound is applied within minutes following injec-
tion. Thus the risk of siRNA degradation due to prolonged
exposure to the in vivo environment is reduced.
Microbubbles however have a number of disadvantages as
delivery agents: they are physically confined to the vascula-
ture and have comparatively poor circulatory stability, typically
1. Introduction
The ability of small interfering RNA (siRNA) to block the trans-
lation of messenger RNA encoding aberrant disease-associated
proteins in a powerful and selective manner provides it with huge
therapeutic potential. Across a wide range of pathologies siRNA
offers a means of specifically knocking down the expression
of the disease causative factor. A prime example of its possible
utility is in the treatment of cancer where degradation of mRNA
encoding antiapoptotic proteins may even enable a synergistic
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exhibiting half-lives of a few minutes.[7] In order to address
these challenges, volatile liquid nanodroplets have been widely
investigated over the past 5–10 years as an alternative means
of promoting ultrasound mediated delivery. Upon exposure to
ultrasound the liquid droplets undergo a phase transition to
form gas microbubbles, but their initial nanoscale size enables
them to both remain in circulation for much longer periods of
time and to extravasate.[8] We have previously demonstrated that
both the stability and phase conversion efficiency of nanodrop-
lets can be considerably improved by the inclusion of iron oxide
nanoparticles.[9] The resulting droplets were able to enhance the
delivery of a conventional chemotherapy drug, paclitaxel, gen-
erating a 40% improvement in cytotoxicity compared with free
drug.
One potential drawback of liquid nanodroplets, however, is
that the phase transition process is associated with large volume
changes and more energetic microbubble activity than that pro-
duced by preformed gas bubbles.[10] Thus there is a potential
risk of collateral damage to the surrounding tissues and also to
the therapeutic material. This is particularly problematic with
molecules such as siRNA that are easily degraded.[11] The aim
of this study therefore was to investigate whether particle sta-
bilized liquid nanodroplets could be used for delivery of siRNA
without impairing functionality.
2. Results and Discussion
2.1. Particle Characteristics
The nanodroplet and nanoparticle formulations used in the
experiments are summarized in Table 1. The mean hydrody-
namic diameter of the chitosan-deoxycholic acid coated per-
fluoropentane nanodroplets (CNDs) (Figure 1a) in deionized
water was 257.6 ± 10.9 nm as determined by dynamic light scat-
tering at 37 °C (Figure 1b). This increased to 3822.2 ± 226.4 nm
when the suspension was exposed to ultrasound (1.8 MHz and
335 kPa peak negative pressure) for 45 s continuously, indi-
cating that the encapsulated perfluoropentane (PFP) under-
went a phase change from liquid to gas. The average diameter
of the chitosan-deoxycholic acid solid particles (CPs) was
170.6 ± 8.9 nm and this changed only slightly to 181.7 nm upon
ultrasound exposure (Figure S5, Supporting Information). The
zeta potential of the CNDs decreased from +57.4 to +40.7 mV
following ultrasound exposure (Figure 1c), corresponding to a
decrease of 29.1% in the surface charge density (please note
that the increase in the surface area of the droplets was much
larger, by an average factor of 219.5). This may be beneficial in
facilitating intracellular uptake of siRNA by decreased electro-
static force after ultrasound treatment.
Figure 1d shows transmission electron microscopy (TEM)
images of CND before and after ultrasound exposure (see also
Figures S2 and S3, Supporting Information). In the left-hand
image, white spots are clearly visible corresponding to PFP
surrounded by darker rings indicating uranyl acetate stained
chitosan-deoxycholic acid shells.[12] Following ultrasound expo-
sure however, these were no longer visible and were replaced
with irregularly shaped particles that were likely produced by
fragmentation of the shell during droplet expansion. There is
an apparent discrepancy between the size of some of the drop-
lets shown in Figure 1d,c prior to ultrasound exposure. This
is likely to be due to vaporization of a portion of the droplets
having occurred in the vacuum environment of the TEM. The
bubbles formed during ultrasound exposure were only stable
when suspended in a 10% glycerol solution (Figure 1c) and so
are not visible in the post ultrasound TEM image. There are
some very small white spots visible in the right-hand panel of
Figure 1d and in Figure S3 (Supporting Information), which
may be droplets that did not vaporize. This is not unexpected
since droplets with diameters of ≈100 nm, will have a much
higher vaporization temperature than particles of 200 nm[12]
and correspondingly the acoustic pressure required to produce
expansion (i.e., the cavitation threshold or Blake pressure[13])
will also be higher.
2.2. Ultrasound Response
Figure 2a, shows bright field optical microscope images of
CNDs captured before and after 45 s of continuous wave (CW)
ultrasound exposure (1.8 MHz and 335 kPa peak negative pres-
sure). Conversion of nanodroplets to microbubbles was readily
observed over this time period. The theoretical size of the
microbubbles, calculated using the ideal gas law, was 2.3 µm
at 37 °C, whereas the experimentally measured average diam-
eter was equal to 6.3 µm.[14] However, the theoretical prediction
did not take into account the additional surface pressure pro-
duced by the shell, coalescence of individual bubbles in close
proximity to each other, or absorption of dissolved gases from
the phosphate buffered saline (PBS).[9,15] Moreover, there may
have been an effect of temperature rise over the course of the
experiment, although the maximum liquid temperature meas-
ured after 3 min of continuous wave ultrasound exposure was
only ≈42 °C (Figure S6, Supporting Information).
In order to detect any changes in the chitosan-deoxycholic
acid shell, CND loaded with Nile red dye were also observed in
the same system under a fluorescent microscope (Figure 2b).
Nile red is lipophilic and so would be expected to label the
strongly hydrophobic deoxycholic acid. Before ultrasound expo-
sure, a collection of small fluorescent objects were visible. After
Table 1. Formulations of nanodroplets and nanoparticles used in the experiments.
Nanodroplets/Materials CND CND/siRNA CND/neg CP CP/siRNA
Core Magnetic nanoparticles (CFe, mmol) 48 48 48 48 48
Perfluoropentane (PFP, µL) 10 10 10 0 0
Shell Chitosan-Deoxycholic acid conjugates (mg) 5 5 5 5 5
siRNA phenotype 0 Cell death Green Fluorescent Protein 0 Cell death
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ultrasound exposure, larger spherical objects with fluorescent
coating could be clearly observed, indicating the formation of
coated microbubbles.
Changes in the concentration of CND between 100 and
1000 nm for different ultrasound exposure times were meas-
ured using a Nanosight system. The concentration of CND
decreased from to 8.99 × 108 to 5.80 × 108 particles mL−1 after
60 s of CW exposure. This reduction indicates that 45% of
CND responded to ultrasound in 1 min, and 57% in 2 min
and then moved out of the measuring range. By contrast, the
change in CP concentration showed an increase of ≈23% at
longer exposure times. This may have been due to the breakup
of CP clusters: the size distribution of CPs also changed after
ultrasound exposure with broadening of the main peak and the
appearance of a second peak at ≈15 nm (Figure S5, Supporting
Information).
2.3. Magnetic Response
As mentioned above, magnetic particles were incorporated into
the droplets to improve vaporization efficiency. They can also facil-
itate droplet localization for targeted delivery. Magnetic retention
of CND was observed in a 127 µm × 50 µm microfluidic channel
into the substrate of which were embedded a small permanent
magnet and an ultrasound transducer.[16] As shown in the micro-
scope images reported in Figure 2d, at a fixed flow rate of 2 mL h−1,
CNDs were concentrated at the wall of the channel closest to the
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Figure 1. a) Schematic representation of chitosan-deoxycholic acid coated perfluoropentane nanodroplets (CNDs). b) Hydrodynamic diameters of
CNDs before (solid line) and after (dotted line) exposure to ultrasound for 45 s in 10% glycerol in water. c) Zeta potential of CNDs before and after
ultrasound exposure. d) TEM images of CND before (left) and after (right) ultrasound exposure for 45 s.
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magnet. After 2 min, ultrasound was applied from the opposite
side of the channel. Microbubbles were continuously generated
from the accumulated droplets and retained in the region by the
magnet. These observations also confirmed successful incorpora-
tion of the iron oxide nanoparticles, whose size (7.5 nm) was too
small to be detected using the available microscopy facilities.
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Figure 2. a) Optical microscope images of CNDs before and after ultrasound exposure for 45 s. Scale bars indicate 200 µm. b) Fluorescent images of
Nile red-loaded CNDs before and after ultrasound exposure for 45 s. Scale bars indicate 200 µm. c) Normalized concentration changes of CNDs and
CPs upon different ultrasound exposure times. d) Accumulation of CNDs at the target site under the influence of a magnetic field and vaporization
by ultrasound exposure. The CNDs were dispersed in PBS, and the flow rate was 0.2 mL h−1. After 2 min of flow, ultrasound was applied from the
opposite side of the channel.
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2.4. siRNA Viability
One concern associated with ultrasound mediated delivery is
the potential for damaging therapeutic molecules. The impact
of ultrasound on the Naked AllStar siRNA and randomly scram-
bled siRNA was therefore investigated at different ultrasound
exposure times (0, 10, 30, 60, and 120 s). Following exposure,
an agarose gel retardation assay was used to detect any siRNA
degradation or structural change (Figure 3a). Electrophoretic
mobility of siRNA in agarose gel was observed, and clear bands
appeared at a same row for all siRNA without drag marks,
which indicated that siRNA did not undergo any significant
degradation under the action of continuous wave ultrasound.
In order to determine whether ultrasound exposure had any
effect upon the gene silencing efficiency of siRNA, the viability
of A549 cells exposed to lipofectamine-siRNA complexes and
ultrasound was compared using the MTS cell proliferation
colorimetric assay. Green fluorescent protein (GFP) siRNA
was used as a negative control for these experiments. After 48
and 72 h incubation, cells treated with the GFP siRNA formu-
lation showed no significant change in viability (Figure 3b).
Conversely, all samples treated with AllStar siRNA underwent
a reduction in cell viability that was more pronounced at 72 h
compared to 48 h, indicating successful gene knockdown. The
ultrasound exposure time had no effect upon the observed
changes in viability.
2.5. siRNA:CND Ratio Optimization
To determine the most effective ratio of AllStar siRNA to CND,
gel electrophoresis was conducted with CND-siRNA complexes
containing different weight ratios of CND to siRNA (Figure 3c).
At a weight ratio of 48, the complexes showed retarded migra-
tion in the gel. Samples were also examined after ultrasound
exposure to assess whether siRNA detachment had occurred.
After 15 min of gel retardation, there was no significant migra-
tion beyond the loading holes, which indicated that either CND
did not release siRNA or it quickly reattached. This is likely to
be beneficial for systemic delivery of siRNA. First, attachment
to a carrier has been shown to stabilize siRNA in the circula-
tion.[17] Second, siRNA does not readily cross cell membranes
through passive diffusion but if it remains attached to frag-
ments of the CND shell this may promote formation of stable
endocytotic vesicles.[18] In addition, if the CND is in sufficiently
close proximity to the target cell then contact area will be greatly
increased during phase transition.
2.6. siRNA Delivery
To determine the gene silencing effect of CND-siRNA com-
plexes and ultrasound exposure, the degree of A549 prolifera-
tion was examined 72 h after treatment. As shown in Figure 4a,
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Figure 3. a) Gel retardation assay of scrambled siRNA and AllStar siRNA exposed to ultrasound at increasing exposure times (0, 10, 30, 60, 120 s).
b) The viability of A549 cells treated with AllStar siRNA-lipofectamine complexes or GFP siRNA-lipofectamine complexes and then incubated for 48 h
(green) and 72 h (red). Prior to treatment, AllStar siRNA was exposed to ultrasound for 0, 10, 30, 60, and 120 s and then complexed with lipofectamine.
c) Complexation test of CND and siRNA in water. Each CND-siRNA complex was prepared at a weight ratio (wr) of CND to siRNA from 0 to 48.
d) Zeta potential values of CND (dashed line) and CND-siRNA (solid line) in water. e) Gel retardation assay of CND-siRNA decomplexation induced
by exposure to ultrasound for different times.
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exposure to the CND-siRNA complexes and ultrasound inhib-
ited the proliferation of cancer cells in a synergistic manner.
A significant reduction in cell viability to 23.4% ± 3.2% was
observed, compared to only 90.3% ± 4.5% from the CND-siRNA
complexes alone and 87% ± 1.47% from CND complexed with
scrambled siRNA and ultrasound.
Solid nanoparticles (CPs) with siRNA produced a negligible
decrease in cell viability in the absence of ultrasound. In com-
bination with ultrasound exposure, they did produce some
reduction in cell viability, potentially as a result of acoustic
streaming and/or radiation force pushing the particles directly
into cells.[19]
2.7. Acoustic Emissions
During ultrasound exposure, the evolution of microbubbles
was monitored using passive acoustic mapping (PAM).[9]
Figure 4a–c shows maps of summed cavitation energy over-
laid on B-mode images from each experiment. When water
was exposed to ultrasound in the absence of any particles,
the maximum PAM signal was 79.26 energy units (please see
the Experimental Section) and the energy distribution was
comparatively uniform. In the absence of any particles but
with cells, a small increase in the PAM signal was detected due
to cavitation at the surface of the A549 cells (max value 239.72
energy units). There was no significant change observed upon
addition of the CP-siRNA complexes. With the CND-siRNA
complexes, however, the maximum PAM signal value was
approximately doubled (572.01 energy units) indicating that
acoustic emissions are well correlated with therapeutic effect
in this system.
2.8. Cell Viability
Apoptosis of A549 cells was also measured 72 h following treat-
ment using annexin V-FITC and propidium iodide (PI) staining
(Figure 4f). Cell shrinkage, changes in DNA content and
changes in the plasma membrane can be observed using flow
cytometry analysis. In early apoptosis, phosphatidyl serine resi-
dues, which have a high affinity for annexin V, are expressed
on the cell surface, and an apoptotic cell can thus be detected.
The results indicate that there were a large number of annexin
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Figure 4. Pre-exposure B-mode images with PAM overlay showing sum of all frames for each exposure, a) for cells only, b) CND/siRNA, and
c) CP/siRNA. Color bars represent cavitation energy (AU). d) Maximum cavitation power (AU) from each frame of PAM data. e) The viability of cells
exposed to ultrasound and incubated for 72 h following treatment with medium, CND/siRNA, CND/negative siRNA, and CP/siRNA shown at an
equivalent siRNA concentration of 20 × 10−9 m. f) In vitro assessment of apoptosis in A549 cells incubated for 72 h following treatment. Flow cytometry
analysis via annexin V-FITC/PI staining was used to observe the induction of apoptosis. Cells in the lower right quadrant are Annexin-positive cells
indicating early apoptotic cells. The cells in the upper right quadrant indicate annexin-positive/PI-positive, late apoptotic cells. g) Annexin V-FITC
analysis for CND/siRNA with ultrasound, CND/siRNA, and CP/siRNA with ultrasound showing degree of apoptosis.
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V-positive cells in the samples exposed to CND-siRNA com-
plexes and ultrasound.
An intense PI signal was observed from the same group.
Interestingly the CP-siRNA complexes produced a slight
increase in the number of late apoptotic cells under ultrasound
exposure. However it was not sufficient to enhance the delivery
efficiency of siRNA and produce a sustained therapeutic effect.
In Figure 4g, annexin V-positive cell populations were indepen-
dently evaluated by flow cytometry. The percentage of cells in
the second peak of each panel indicates the total percentage of
apoptotic cells.[20] This analysis revealed that the CND-siRNA
complexes had a significant effect on inducing cancer cell apop-
tosis (52.4%) in combination with ultrasound exposure.
2.9. Cell Uptake
Confocal scanning laser microscopy was used to observe the
cellular uptake of fluorescent siRNA carried by nanodroplets
to GFP-expressing MCF7 cells (Figure 5). After 5 min of incu-
bation without ultrasound exposure, low intensity red fluores-
cence was detected in the cells incubated with CND-siRNA
complexes. This was significantly increased by ultrasound expo-
sure even with thorough washing after treatment. Furthermore,
widespread distribution of red fluorescent siRNA was achieved
by ultrasound stimulation. Ultrasound exposure was not found
to promote delivery of free siRNA, i.e., in the absence of CNDs.
3. Conclusion
The aim of this study was to investigate particle stabilized
liquid nanodroplets as a vehicle for ultrasound mediated
delivery of siRNA. Complexes consisting of chitosan-deoxy-
cholic acid nano particles containing perfluoropentane and iron
oxide were successfully prepared and conjugated to cell death
control siRNA. The complexes were found to be stable at 37 °C
for up to 4 h in serum (Figure S4, Supporting Information).
Breast cancer cells and lung cancer cells were exposed to ultra-
sound (500 kHz, 1 MPa peak-to-peak focal pressure, 40 cycles
per burst, 1 kHz pulse repetition frequency, 10 s duration) in
the presence of the complexes or similar particles that did not
contain PFP. We verified that the functionality of siRNA was
not adversely affected by the treatment protocol and identi-
fied the optimal ratio of siRNA to nanodroplets. We also con-
firmed the potential for magnetic localization of the complexes
using an externally applied magnetic field. Both delivery and
gene silencing were successfully demonstrated, with a fourfold
reduction in cell viability being produced by
the CND-siRNA complexes compared to the
control group. Monitoring of acoustic emis-
sions throughout the ultrasound exposure
period indicated that there was a positive cor-
relation between the energy of these emis-
sions and treatment efficacy.
4. Experimental Section
Materials: Deoxycholic acid, chitosan
oligosacharide, N-hydroxysuccinimide (NHS),
heparin sodium salt from porcine intestinal
mucosa (heparin), dimethyl sulfoxide (DMSO), gel-
loading buffer, and tris-borate-EDTA (TBE) buffer
were purchased from Sigma-Aldrich (Dorset, UK).
AllStars HS cell death control siRNA and scrambled
siRNA was ordered from Qiagen (Manchester, UK).
Perfluoropentane (99%) was obtained from Apollo
Scientific (Cheshire, UK). Silencer GFP siRNA,
Lipofectamine 2000, fetal bovine serum (FBS),
PBS, and Dulbecco’s modified Eagle’s medium
(DMEM) were obtained from Life Technologies Ltd
(Paisley, UK). Annexin V-FITC, PI, and nuclease-free
water was purchased from ThermoFisher Scientific
(Bicester, UK). Iron oxide (Fe2O3) nanoparticles
coated with oleic acid were synthesized using
capillary microfluidic devices (d0 = 7.5 ± 0.35 nm,
CFe = 0.48 mol L−1) as described in previous work.[21]
All chemicals were of reagent grade and used
without further purification.
Nanoparticle Fabrication: In order to enable
siRNA attachment to the nanodroplet surface,
it was necessary to modify the formulation from
that previously published.[9] Amphiphilic chitosan
oligosaccharide and deoxycolic acid (COSD) were
synthesized by a coupling reaction of succinimido
deoxycholic acids to the primary amine group
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Figure 5. Confocal microscope images of the Cy3-labeled siRNA using CND/siRNA complex or
free siRNA with MCF 7 cells expressing GFP after 5 min of incubation. One of the cell chambers
treated with CND/siRNA complexes was exposed to ultrasound after incubation (500 kHz,
1 MPa peak-to-peak focal pressure, 40 cycles per burst, 1 kHz PRF, 10 s duration). The last
column shows merged images from first and second columns.
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of chitosan chains[22] (Figure S1, Supporting Information). COSD
nanoparticles (CPs) incorporating magnetic nanoparticles in their
core were prepared by an oil-in-water emulsification method. COSD
nanodroplets (CNDs) were then generated by sonication of CPs in the
presence of PFP. Deoxycholic acid is strongly hydrophobic and thus PFP
can be stabilized in the particle core. The chitosan amino groups impart
a positive charge to both the CPs and CNDs surfaces enabling siRNA to
be easily bound through electrostatic interactions (Figure 1a).
Deoxycholic acid (200 mg, 0.5 mmol) and NHS (76 mg, 0.67 mmol)
were dissolved in anhydrous tetrahydrofuran (20 mL). Following the
addition of 1,3-dicyclohexylcarbodiimide (136 mg, 0.67 mmol), the
solution was stirred at 4 °C for 6 h. The urea byproducts were removed
by filtration; the filtrate was poured into cold n-hexane (120 mL), and
then the precipitates were dried in vacuum overnight. The prepared
succinimido deoxycholates were reacted with the primary amine groups
of chitosan oligosaccharide (COS) via the carbodiimide couple reaction,
forming chitosan grafted with deoxycholates (COSD). COS (80 mg) was
dissolved in a 9/1 (v/v) mixture of DMSO and deionized water, and then
succinimido deoxycholates (49.8 mg) were added to the solution. The
reaction mixtures were magnetically stirred for 12 h at room temperature.
The resulting solutions were then precipitated into the excess amount of
acetone. The precipitates were recovered by centrifugation at 2000 rpm
for 5 min, washed with acetone twice and dried under vacuum.
After dispersion of the dried COSD in distilled water, the suspension
(5 mg mL−1) was sonicated with an ultrasonic cell disruptor (XL 2000,
Misonix Inc. Farmingdale, NY, USA) for 1 min. The prepared suspension
was filtered through a 1.0 µm pore syringe filter to remove large
aggregates; then 100 µL of the iron oxide nanoparticles in chloroform
was added to the filtered suspension. The mixture was sonicated for 30 s
and the solvent then evaporated with stirring. The resulting suspension
of COSD nanoparticles (CP) was sonicated for a further 10 s with
perfluoropentane to prepare COSD nanodroplets.
Nanoparticle Characterization: The hydrodynamic diameters of the
prepared CPs and CNDs were measured using a zeta-potential and
particle size analyzer (Malvern Instruments, UK) in triplicate at 37 °C.
The concentration of nanodroplets dispersed in water was measured
by nanoparticle tracking analysis (NanoSight, UK). The size and shape
of the particles were determined using bright field TEM (FEI Tecnai
12, USA). 10 µL of each suspension was dispersed in aqueous solution
(2 mg mL−1) and deposited on a Formvar/carbon-supported copper grid.
The grid was dried in air for 1 h at room temperature and samples were
stained with 2% (w/v) of uranyl acetate solution before examination to
enable visualization of the polymer.
siRNA Attachment: Cell death control siRNA was used to determine
any synergistic effect on the inhibition of cell proliferation with
ultrasound. siRNA-CND complexes were prepared with different weight
ratios (wr) of CND to siRNA from 3 to 48. siRNA (13 µg, 1 nmol)
dissolved in nuclease-free water was mixed with CND (wr 39, 78,
156, 312, and 624) diluted with nuclease-free water. The mixture was
incubated at room temperature for 20 min. The apparent zeta potential
of both CND and siRNA-CND complexes was measured using the same
zeta-potential and particle size analyzer (Malvern Instruments, UK) at
20 °C. To confirm binding of siRNA to the CND, electrophoresis (1%
agarose gel) was carried out at 110 V for 15 min in TBE buffer. The band
was stained with ethidium bromide (EtBr) included in the agarose gel.
Acoustic Response: A multilayered acoustic resonator with an optically
transparent chamber was used to compare the performance of different
nanodroplet formulations, in terms of their phase transition efficiency
and drug release upon exposure to ultrasound. Details of this device
have been published previously[9] but briefly the resonator consists of a
piezoelectric transducer, a carrier layer which couples the acoustic energy
to the other components of the device, a fluid layer with nanodroplets
in suspension, and a reflector layer which reflects the acoustic energy
back into the device. The experimentally measured resonance frequency
for the device was ≈1.85 MHz, in agreement with computational
predictions. The transducer was driven with a continuous wave at a
fixed peak-to-peak voltage of 40 V. The resulting peak rarefactional
pressure in the chamber was 335 kPa, measured with a calibrated fiber
optic hydrophone (Precision Acoustics, Dorchester, UK). To observe the
response of CNDs to ultrasound 400 µL of the prepared suspension was
pipetted into the device and covered using a glass slide. The device was
then mounted on the stage of Nikon TI Eclipse fluorescent microscope
(Nikon UK Ltd, Kingston upon Thames, UK) and the solution exposed
to ultrasound for the period of interest (between 0 and 90 s) at room
temperature.
Magnetic Response: To observe magnetically guided accumulation
and in situ vaporization, a cubic neodymium cross-section permanent
magnet (NdFeB, N52, 12 mm x 12 mm × 12 mm, CMS Magnetics)
and piezoelectric transducer (14 mm × 15 mm × 2 mm, PZ26, Meggit
PLC, UK) were assembled in a microfluidic device comprising of
a 127 µm × 50 µm (width × thickness) straight microchannel. The
microchannel was fabricated using a replica molding microfabrication
technique.[16] The CND suspension was flown through the channel at a
flow rate 0.2 mL h−1 controlled using a syringe pump (NE-1000 New Era
Pump Systems, Inc, Hertfordshire, UK). Continuous wave ultrasound
was applied during the flow (1.85 MHz, peak rarefactional pressure of
335 kPa, peak-to-peak voltage of 40 V). Accumulation and vaporization
of nanodroplets were observed under an optical microscope. Images
were acquired with a Nikon Eclipse Ti microscope (Nikon Instruments
Europe B.V., Amsterdam, The Netherlands).
siRNA Viability: Free siRNA in DEPC aqueous suspension was exposed
to ultrasound using the device described above for periods ranging
from 0 to 120 s (1.85 MHz, peak rarefactional pressure of 335 kPa,
peak-to-peak voltage of 40 V). Electrophoresis (1% agarose gel) was
then carried out at 110 V for 15 min in TBE buffer to determine whether
or not the siRNA had been structurally damaged. The band was stained
with EtBr included in the agarose gel. The viability of A549 cells (human
lung cancer cells) was also evaluated to determine whether the gene
silencing activity of the siRNA had been affected by ultrasound exposure.
A549 cells were seeded at a 96-well plate at a density of 1 × 104 cells
per well and grown in DMEM supplemented with 10% FBS at 37 °C
for 24 h. The culture medium was replaced with DMEM containing
20 × 10−9 m siRNA-lipofectamine complexes, and further incubated for
48 or 72 h at 37 °C. The number of viable cells was determined using the
MTS colorimetric assay (Promega UK, Southampton, UK). Next, CND–
siRNA complexes were exposed to ultrasound with the same conditions
for 0, 30, 60, and 180 s and subjected to gel electrophoresis to assess
release of siRNA.
Cell Viability: The cytotoxicity of the nanoparticles and nanodroplets
was evaluated by examining the inhibition of cancer cell proliferation. The
relevant suspensions were diluted with PBS to give a final concentration
of siRNA of 20 × 10−9 m (where relevant) based on the binding efficiency
determined via electrophoresis as above and the relative concentrations
of siRNA and CND/CP in the suspension. A549 cells were seeded
in Ibidi cell dishes at a density of 1 × 105 cells per dish and grown in
DMEM at 37 °C for 24 h. The culture medium was replaced with serum-
free DMEM containing one of the following: (i) CND with cell death
control siRNA (CND/siRNA), (ii) CND/GFP siRNA (CND/neg), and (iii)
nanoparticles with cell death control siRNA (CP/siRNA). The cell culture
dishes were sealed using acoustically compatible polydimethylsiloxane
(PDMS) lids.[23] Devices were mounted on top of a permanent magnet
Halbach array.[24] The cells were incubated at 37 °C for 5 min before
treatment.
For ultrasound exposure, the assembly was aligned with the focus
of a 500 kHz single-element focused ultrasound transducer (model
H-107B-10; Sonic Concepts, USA) in a tank containing degassed,
deionized water at 37 °C. The transducer featured a rectangular cutout
through which an imaging linear array (model L11-4v; Verasonics Inc.,
USA) was aligned.[25] Samples were exposed to 10 s of ultrasound
(pulse center frequency of 500 kHz, 1 MPa peak-to-peak focal pressure,
40 cycles per burst, 1 kHz pulse repetition frequency[26]) at five points
3 mm apart, while the imaging array passively recorded the acoustic
emissions from every 10th burst for the first second of each exposure
(100 frames, 128 channels, 170 µs per run) to an ultrasound research
platform (Verasonics V1; Verasonics Inc., USA) for subsequent
analysis.
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After ultrasound exposure, cells were further incubated at 37 °C
for 24 h. The number of viable cells was determined using the MTS
colorimetric assay. To assess apoptosis, treated A549 cells were stained
with annexin V-FITC and PI solution. The cells were collected by
centrifuging at 1100 rpm for 5 min after treatment and washed twice
in PBS. Washed cells were suspended in a binding buffer and then
annexin V-FITC and PI solution were added. The cell solution was gently
vortexed and incubated for 15 min at room temperature in the dark.
The fluorescence intensities of annexin V-FITC and PI were analyzed by
flow cytometry (FACSort, BD Bioscience, Oxford, UK). Cell populations
containing annexin V-FITC and PI were analyzed to determine the
numbers of early/late apoptotic and dead cells.
MCF-7 cells (human breast cancer cells) expressing GFP were seeded
in Ibidi cell dishes at a density of 5 × 104 cells per dish and incubated for
24 h with DMEM supplemented with 10% FBS. Cy3 labeled siRNA-CND
complexes was prepared to compare the level of cellular uptake
promoted by different ultrasound exposure conditions. The culture
medium was replaced with Opti-MEM to which were added either CND-
siRNA complexes or free siRNA of 10 × 10−9 m. The cell culture dishes
were then sealed using PDMS lids, mounted on top of a permanent
magnet Halbach array and exposed to ultrasound as described above.
After treatment with one of the siRNA formulations and/or ultrasound,
cells were washed with PBS three times and fixed with 4% formaldehyde
solution in PBS. The cells were then observed in the Ibidi chamber
by confocal microscopy (Zeiss780, Zeiss, Cambridge, UK). Images
were collected using 488 and 561 nm laser lines for excitation and a
20× objective.
Cavitation Monitoring: The acoustic emissions captured by the
imaging array for each experiment were high-pass filtered at 1 MHz
to remove the main drive signal and mapped in space to provide an
estimate of the acoustic power of cavitation emissions using the
reconstruction algorithm described in ref. [27] Acoustic maps over
a 20 × 20 mm area about the ultrasound focus were generated for
each frame of the received data. The sum of these maps from each
experiment was used to estimate the total energy of acoustic emissions,
and overlaid on pre-exposure B-mode images to indicate their spatial
distribution. In addition, the maximum value in the maps for each
individual frame was used to evaluate the evolution of cavitation activity
over time.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author (doi.10.5287/bodleian:QyZaRYqA1).
Acknowledgements
The authors would like to thank James Fisk and David Salisbury for
construction of the transducer and phantom holders used in this study.
Calum Crake acknowledges the support of the RCUK Digital Economy
Programme Grant No. EP/G036861/1 (Oxford Centre for Doctoral
Training in Healthcare Innovation). In addition, the authors are grateful
to the EPSRC for supporting this research through Grant No. EP/
I021795/1 and Programme Grant No. EP/L024012/1 (OxCD3: Oxford
Centre for Drug Delivery Devices).
Received: November 6, 2016
Revised: January 7, 2017
Published online:
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