Page 1
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7678
July 13, 2015
C2015 American Chemical Society
Piezoelectric Nanoparticle-Assisted
Wireless Neuronal Stimulation
Attilio Marino,*,†,‡Satoshi Arai,§Yanyan Hou,§Edoardo Sinibaldi,†Mario Pellegrino,^Young-Tae Chang,),#
Barbara Mazzolai,†Virgilio Mattoli,†Madoka Suzuki,*,§,rand Gianni Ciofani*,†
†Center for Micro-BioRobotics, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025 Pontedera (Pisa), Italy,‡The Biorobotics Institute, Scuola Superiore
Sant'Anna,VialeRinaldoPiaggio34,56025Pontedera(Pisa),Italy,§WASEDABioscienceResearchInstituteinSingapore(WABIOS),BiopolisWay11,#05-02Helios,
138667 Singapore,^Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, University of Pisa, Via Savi 10, 56126 Pisa, Italy,
)
Department of Chemistry, National University of Singapore, MedChem Program of Life Sciences Institute, National University of Singapore, 3 Science Drive 3,
117543 Singapore,#Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR),
Biopolis,138667Singapore,andrOrganizationforUniversityResearchInitiatives,WasedaUniversity,#304,Block120-4,513Waseda-Tsurumaki-Cho,Shinjuku-Ku,
162-0041 Tokyo, Japan
T
nervous system,1and several innovative
techniques are being explored in order to
address this challenge. Among these, the
rapid development of genetically encoded
actuators/sensors, i.e., optogenetics,2is al-
lowing for the detailed investigation of the
mechanisms of various diseases, especially
those presenting a neuronal etiology, in
different animal models, thanks to a fine
cellactivitymodulation/monitoring.3?7How-
ever, the exploitation of optogenetics is
currently limited by several complications,
including the phototoxicity8and the scarce
penetration of the light through the tissues
due to their opacity at the suitable wave-
lengths.9Other well-characterized neural
stimulation approaches are represented
by the deep brain stimulation (DBS),10the
trans-cranialdirect
(tDCS),11,12and the trans-cranial magnetic
stimulation (TMS).13Drawbacks of DBS are
he possibility to stimulate/modulate
the neural activity is often limited
by the restricted accessibility of the
current stimulation
thenecessityofaninvasivesurgicaloperation,
followed by inflammation and gliosis at the
implant site, while the main disadvantage of
thetDCSandTMSisthelowspatialresolution
(a brain volume of about 1 cm in diameter).
Ultrasounds(US)canbeinsteadexploited
for trans-cranial stimulation without the re-
quirement of surgical processes, and with a
resolution of about 3 mm,14which can be
further improved by the use of hyperlenses
and acoustic metamaterials.15,16However,
the heterogeneous results obtained with the
USstimulation,thatincertainconditionsmay
induceneuralsilencingratherthanexcitation,
are influenced by the physical parameters of
the US, including frequency and power. Re-
cently, Tufail et al. reported an exhaustive
summary of the US-mediated neuromodula-
tion effects observed by using different US
parameters and different neural models.17
US can be exploited in combination with
piezoelectric materials in order to generate
direct-current output:18,19indeed, piezo-
electric materials are able to efficiently
*Address correspondence to
attilio.marino@iit.it,
suzu_mado@aoni.waseda.jp,
gianni.ciofani@iit.it.
Received for review May 26, 2015
and accepted July 13, 2015.
Published online
10.1021/acsnano.5b03162
ABSTRACT Tetragonal barium titanate nanoparticles (BTNPs) have been
exploitedasnanotransducersowingtotheirpiezoelectricproperties,inorderto
provide indirect electrical stimulation to SH-SY5Y neuron-like cells. Following
application of ultrasounds to cells treated with BTNPs, fluorescence imaging of
iondynamicsrevealedthatthesynergicstimulationisabletoelicitasignificant
cellular response in terms of calcium and sodium fluxes; moreover, tests with
appropriateblockersdemonstratedthatvoltage-gatedmembranechannelsare
activated. The hypothesis of piezoelectric stimulation of neuron-like cells was
supported by lack of cellular response in the presence of cubic nonpiezoelectric
BTNPs, and further corroborated by a simple electroelastic model of a BTNP subjected to ultrasounds, according to which the generated voltage is
compatible with the values required for the activation of voltage-sensitive channels.
KEYWORDS: barium titanate nanoparticles.ultrasounds.piezoelectricity.SH-SY5Y cells.calcium imaging
ARTICLE
Page 2
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7679
generate electricity in response to the US mechanical
stimulations. Taking advantage of the piezoelectricity
of different nanomaterials, such as ZnO nanowires and
BaTiO3nanoparticles (BTNPs), it is possible to obtain
US-driven nanogenerators able to generate output
currents inside biological liquid for energy harvesting
andforself-poweredelectronics.20?22Inapreviouswork
of our group, the US-driven piezoelectric stimulation of
PC12neural-likecellswasperformedforthefirsttimeby
using boron nitride nanotubes (BNNTs).23In this work, a
significant enhancement of the neurite outgrowth was
observed in response to the combined US þ BNNT
stimulus, with respect to the US stimulus without the
presence of the piezoelectric nanoparticles; moreover,
the significant increase of the neurite length dependent
by the US þ BNNT stimulation was suggested to be
mediated bya Ca2þinflux. However, theimpossibilityto
perform electrophysiological recordings in conjunction
withtheUSstimulationhasnotallowedthemechanisms
giving rise the Ca2þinflux (i.e., piezoelectric-mediated
plasma membrane depolarization) to be investigated,
and consequently the different ion currents involved to
be elucidated.
In this work, we carried out neural stimulation with
US and piezoelectric BTNPs characterized by a tetra-
gonal crystalline configuration, and the effects of both
US and US þ BTNP stimulations on SH-SY5Y-derived
neurons were deeply investigated, by exploiting ima-
gingtechniquesfordetectingthe Ca2þ/Naþfluxesand
temperature levels. Our results show as the stimula-
tion with US þ piezoelectric BTNPs was able to induce
tetrodotoxin (TTX) and cadmium (Cd2þ) sensitive
high-amplitude Ca2þtransients, and that the observed
transients are specifically evoked thanks to the piezo-
electric properties of these nanoparticles with single
cell resolution. Our findings thus strongly support the
hypothesis that the piezoelectric stimulation is able
to induce a Ca2þinflux, which likely mediates the
enhancement of the neurite outgrowth and neural
differentiation,23,24and suggest the possible use of
this approach as a wireless tool to modulate neuronal
activity evendeeply in vivo,by acombination of piezo-
electric nanoparticles and a pulse of ultrasounds.
RESULTS
Tetragonal Barium Titanate Nanoparticle Characterization.
Images of the BTNPs used in this work, wrapped with
gum Arabic, are provided in Figure 1a (scanning
electron microscopy, SEM) and Figure 1b (trans-
missionelectronmicroscopy,TEM).Theseobservations
revealed quite well-dispersed structures, with some
aggregates of a few nanoparticles. The hydrodynamic
size of the BTNPs, measured through dynamic light
scattering (DLS), resulted of 479.0 ( 145.3 nm, with a
polydispersity index of 0.180. The Z-potential was
?40.4 ( 5.2 mV, highlighting an excellent stability of
thedispersion.HydrodynamicsizeandZ-potentialwere
evaluated also in experimental conditions (50 μg/mL of
BTNPs in artificial cerebrospinal fluid) and resulted
573.3 ( 173.8 nm and ?17.2 ( 1.2 mV, respectively.
Most useful for tracking in cell experiments, BTNPs
resulted visible through confocal fluorescence
imaging (Figure 1c), by using an excitation at 633 nm
and a collection from 645 to 745 nm. From XRD anal-
ysis (Figure 1d), BTNPs resulted to have a perovskite-
like crystallographic structure. Tetragonal phase of
Figure 1. Barium titanate nanoparticles (BTNPs) characterized by a tetragonal crystalline structure. SEM (a), TEM (b) and
confocal fluorescence (c) imaging of BTNPs. In (c), confocal fluorescence image (red) is merged with the transmitted light
image (gray) of the same field of view. The crystallographic structure revealed thanks to the XRD analysis shows two close
peaks at 2θ = 44.85? and 2θ = 45.38?, specific of the tetragonal configuration of the crystal (d).
ARTICLE
Page 3
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7680
BaTiO3, typical of piezoelectric nanoparticles, was
detected, with two close peaks at 2θ = 44.85? and
2θ = 45.38?.25
US-Driven BTNP Stimulation Induces Ca2þTransients. In
order to evaluate the BTNPs/neurons interaction,
confocal fluorescence microscopy was carried out
(Figure 2). Particularly, high amount of BTNPs (in red)
was observed associated with the plasma membrane
(in green) of the SH-SY5Y-derived neurons after 24 h
from the BTNP treatment (Figure 2a); Figure 2bdepicts
the 3D rendering of a confocal z-stack acquisition (see
also Video S1, provided as Supporting Information).
From all these observations, we can conclude as
nanoparticles are mainly associated with the plasma
membrane without a significant cellular internaliza-
tion. Finally, BTNPs were observed associated not only
to the membrane of the SH-SY5Y cell bodies, but also
to their neurites, as shown by Figure 2c.
Cellular viability following the experimental proce-
dureswasconfirmedbyWST-1metabolicactivityassay
and propidium iodidestaining, thelatter evaluating the
integrityofthecellularmembrane after thestimulation.
Four experimental groups were considered: control
cultures, cultures treated with BTNPs (50 μg/mL), cultures
treated with US (0.8 W/cm2), and cultures treated with
bothBTNPs(50μg/mL)andUS (0.8W/cm2).Resultsare
provided in Figure S1 as Supporting Information, and
highlight no statistically significant differences among
the different treatments in terms of both metabolic
activity (Figure S1a, evaluated after 24 h since the
stimulation) and membrane integrity (Figure S1b,
evaluated immediately after the stimulation in order
to highlight also temporarily phenomena of mem-
brane permeabilization).
After the evaluation of the BTNP/cell interactions
at the level of the plasma membrane, we monitored
the intracellular Ca2þdynamics in response to the
US stimulation performed at different intensities (0.1, 0.2,
0.4 and 0.8 W/cm2), with or without BNTPs (Figure 3).
Interestingly, we observed that, by stimulating
SH-SY5Y-derived neurons with BTNPs þ US at 0.8 W/cm2,
it was possible to activate high-amplitude Ca2þtran-
sients (ΔF/F0peak = 0.62 ( 0.12), significantly higher
with respect to the low-amplitude Ca2þtransients
Figure 2. Confocal fluorescence microscopy of BTNPs associating to the neuronal plasma membranes. (a) Characteristic
confocalz-stackofBTNP-treatedSH-SY5Y-derivedneurons(neuronalplasmamembranesingreen,BTNPsinredandnucleiin
blue).(b)3D renderingof theconfocalz-stacksof thesamefieldas in(a).BTNPswerealsodetectedassociatingtotheneurite
membranes (c).
ARTICLE
Page 4
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7681
detectedinalltheotherconditions(p<0.05),including
the treatment with US at 0.8W/cm2but without BTNPs
(ΔF/F0peak=0.15(0.04;pleaserefertotheTable1for
the comparison of all of the peak amplitudes). Concern-
ing the tests performed with US intensities < 0.8 W/cm2,
nosignificantdifferencesintermsofcalciumtransients
amplitude evoked by US þ BTNPs were found with
respect to the plain US stimulation (p > 0.05). In the
following results, relative to the investigation of the
mechanismsatthebaseoftheobservedphenomenon,
justthestimulationatthehighestintensity(0.8W/cm2)
willbethusconsidered.VideoS2,providedasSupporting
Information, shows the Ca2þimaging time-lapse course
(18X accelerated) performed on cultures stimulated
with US (0.8 W/cm2) in the presence of BTNPs. The US
stimulation was applied at tvideo= 1 s.
In order to investigate the ion channels involved in
theobservedCa2þtransients,Ca2þimagingexperiments
wereperformedinthepresenceofblockerseitherofthe
voltage-gated Ca2þchannels (Cd2þ) or of the voltage-
gatedNaþchannels(TTX)(Figure4).26,27Figure4a?dare
representative ΔF/F0traces relative to Ca2þimaging
time-lapses of neurons stimulated by US, US þ BTNPs,
Cd2þþUSþBTNPs,andTTXþUSþBTNPs,respectively;
Figure 3. Calcium imaging of SH-SY5Y-derived neurons in response to the US stimulation performed at different intensities
(0.1, 0.2, 0.4 and 0.8 W/cm2), with or without BNTPs: time courses of the ΔF/F0traces. Arrows indicate the moment when the
5-sUSpulsewasinitiated;intheinletof eachgrapharepresentativecalciumimagingtime-lapseframeisreported(att=30).
ARTICLE
Page 5
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7682
in the inlet of each graph a representative Ca2þimaging
time-lapse frame is reported (more time-lapse images
are reported in Figure S2 as Supporting Information).
In Figure 4a we can appreciate low-amplitude Ca2þ
transients (ΔF/F0 peak = 0.15 ( 0.04) induced by
the US stimulus. In Figure 4b, the US in the pres-
ence of BTNPs induce significantly higher Ca2þpeaks
(ΔF/F0peak = 0.62 ( 0.12; p < 0.05) compared to the
US stimulation without nanoparticles. These high-
amplitude Ca2þtransients were suppressed by both
theCd2þ(Figure4c)andtheTTX(Figure4d)treatments.
Inbothconditions(Cd2þþUSþBTNPsandTTXþUSþ
BTNPs) we detected low-amplitude Ca2þpeaks (ΔF/F0
peak = 0.16 ( 0.02 in the presence of Cd2þand ΔF/F0
peak = 0.14 ( 0.01 in the presence of TTX), similar to
those observed by stimulating with US without BTNPs
(ΔF/F0peak = 0.15 ( 0.04; p > 0.05; Table 1), thus
suggesting that the induction of the high-amplitude
Ca2þtransients by the US þ BTNPs stimulation is medi-
ated by both Ca2þand Naþvoltage-gated channels.
US-Driven BTNP Stimulation Induces TTX-Sensitive NaþTran-
sients. Possible effects of the stimulation on the Naþ
fluxes were evaluated by performing Naþimaging
experimentsonSH-SY5Y-derivedneuronstreatedwith
US, US þ BTNPs, Cd2þþ US þ BTNPs, and TTX þ US þ
BTNPs. Representative ΔF/F0traces relative to Naþ
imaging time-lapses are shown in Figure 5a?d; in the
inletofeachgrapharepresentativeNaþimagingtime-
lapse frame is reported (more time-lapse images
are reported in Figure S3 as Supporting Information).
The US stimulation without BTNPs was not able to
induce any appreciable Naþpeak (Figure 5a), while
aNaþtransient wasclearlydetected inresponsetothe
US þ BTNP activation (ΔF/F0peak = 0.032 ( 0.001;
Figure 5). Naþpeaks of lower amplitude were revealed
by stimulating SH-SY5Y-derived neurons with US þ
BTNPs in the presence of the voltage-gated Ca2þchan-
nelblockerCd2þ(ΔF/F0peaks=0.011(0.001;Figure5c),
TABLE 1. Calcium/Sodium Transient Amplitude (In Terms
of ΔF/F0Peak Average ( Standard Error) Measured in
Response to the Different Stimulations and Treatments
calcium imaging
treatmentUSUS þ BTNPs
0.14 ( 0.03
0.15 ( 0.02
0.22 ( 0.04
0.62 ( 0.12
0.16 ( 0.02
0.14 ( 0.01
0.12 ( 0.03
no transients
0.78 ( 0.24
0.12 ( 0.01
0.1 W/cm2
0.2 W/cm2
0.4 W/cm2
0.8 W/cm2
0.8 W/cm2
No transients
0.09 ( 0.02
0.16 ( 0.02
0.15 ( 0.04
N/A
N/A
0.16 ( 0.04
no transients
0.15 ( 0.05
0.15 ( 0.04
Cd2þ
TTX
EGTA
thapsigargin þ EGTA
gentamicin
cubic crystal
sodium imaging
treatmentUSUS þ BTNPs
0.032 ( 0.001
0.011 ( 0.001
no transients
0.8 W/cm2
0.8 W/cm2
no transients
N/A
N/A
Cd2þ
TTX
Figure 4. US þ BTNP stimulation (0.8 W/cm2) evokes Cd2þand TTX-sensitive calcium transients. Representative ΔF/F0traces
relative to calcium imagingtime-lapses of SH-SY5Y-derived neuronsstimulated by US (a), US þ BTNPs (b), US þ BTNPs in the
presence of Cd2þ(c) or TTX (d). Arrows indicate the moment when the 5-s US pulse was initiated; in the inlet of each graph a
representative calcium imaging time-lapse frame is reported (at t = 50).
ARTICLE
Page 6
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7683
while no peaks were detected in the TTX treatment
(Figure 5d).
InvestigationofCa2þSourcesInvolvedintheUS-DrivenBTNP
Stimulation. The Ca2þsources involved in the stimula-
tions, e.g., intracellular stores and/or extracellular envi-
ronment, were investigated as reported in Figure 6 (in
the inlet of each graph a representative Ca2þimaging
time-lapse frame is reported; more time-lapse images
are reported in Figure S4 as Supporting Information).
The US stimulation in Ca2þ-free conditions (and in the
presence of 5 mM of the Ca2þchelator ethylene glycol
tetraacetic acid, EGTA) induced low-amplitude Ca2þ
transients (ΔF/F0peak = 0.16 ( 0.04; Figure 6a), non-
significantly different from those observed in standard
conditions, i.e., in the presence of 2 mM extracellular
Ca2þ(ΔF/F0peak=0.15(0.04;p>0.05;Figure4a).This
result suggests that the intracellular Ca2þstores are
implicated in the low-amplitude Ca2þtransients ob-
served during the stimulations with US, but not in the
presence of BTNPs. Interestingly, we also observed
low-amplitude Ca2þtransients when stimulating with
BTNPs and US in the EGTA-supplemented Ca2þ-free
medium(ΔF/F0peak=0.12(0.03;Figure6b).Thislow-
amplitude peak is significantly lower compared to the
high amplitude peak measured in standard conditions
(i.e., 2 mM of extracellular Ca2þ; ΔF/F0peak = 0.62 (
0.12; p < 0.05; Table1), suggesting that the US þ BTNP
stimulation, conversely to the plain US stimulation,
is able to activate the Ca2þinflux through the plasma
membrane. This result is in agreement with the pre-
vious observation highlighting that the high-ampli-
tude peaks are inhibited by the treatments with TTX
and Cd2þ, which are blockers of the cell membrane
voltage-gated channels.26,27Further study suggests
that the source of these low-amplitude Ca2þtransi-
ents is the endoplasmic reticulum (ER): both the
low-amplitude peaks reported after the plain US
(Figure 6c) and the US þ BTNP (Figure 6d) stimulation
in extracellular Ca2þ-free conditions completely
disappear by depleting the Ca2þflux from ER with
thapsigargin, suggesting that the ER represents the
involved Ca2þstore.
Thermal Effects of the US-Driven BTNP Stimulation. US
stimulations higher than 0.5 W/cm2are known to
locally increase the temperature,17,28,29and the ER is
prone to release Ca2þin response to a heat pulse.30,31
We therefore investigated whether the US and US þ
BTNP stimuli are able to induce a temperature incre-
ment at ER level (Figure S5, supplied as Supporting
Information). In order to monitor the ER tempera-
ture during the stimulations, we took advantage of a
thermosensitive fluorescent dye able to specifically
target the ER, the ER thermo yellow.32In Figure S5a
wecanappreciatethecolocalizationofthefluorescence
Figure5. USþBTNPstimulation(0.8W/cm2)inducesTTX-sensitivesodiumtransients.RepresentativeΔF/F0tracesrelativeto
sodiumimagingtime-lapsesofSH-SY5Y-derivedneuronsstimulatedbyUS(a),USþBTNPs(b),USþBTNPsinthepresenceof
Cd2þ(c)orTTX(d).Arrowsindicatethemomentwhenthe5-sUSpulsewasinitiated;intheinletofeachgrapharepresentative
sodium imaging time-lapse frame is reported (at t = 27).
ARTICLE
Page 7
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7684
signal stemming from the ER thermo yellow with that
one of the ER tracker, demonstrating the high specifi-
city of the ER thermo yellow in SH-SY5Y cells. The
sensitivity of the ER thermo yellow in SH-SY5Y cells
was determined to be ?2.0%/?C by linearly fitting
the F/F0data measured at different ER temperature
increment (ΔT, ?C) starting from the room tem-
perature (Figure S5b). Figure S5c and S5d show
representative time courses of the ER thermometer
fluorescence signal during the stimulation with US
(0.8 W/cm2) and with US þ BTNPs, respectively. The
relativeΔT(?C)tracesareshowninFigureS5eandS5f,
showingthattheUSstimulationisabletoincreasethe
ER temperature both in presence (1.66 ( 0.30 ?C) and
in absence (1.68 ( 0.31 ?C) of BTNPs, but without
anysignificantdifferencebetweenthetwoconditions
(p > 0.05).
Further Evaluations of the Mechanisms at the Base of the
US-DrivenBTNPStimulation. Theroleofmechanosensitive
channels in the low-amplitude and high-amplitude
Ca2þtransients respectively observed after the US
and US þ BTNP stimulations was investigated by per-
foming Ca2þimaging experiments in the presence of
200 μM gentamicin, a blocker of mechano-sensitive
cation channels that does not affect the voltage-gated
Ca2þcurrents.33?35Eveninthepresenceofgentamicin,
low-amplitude (ΔF/F0peak = 0.15 ( 0.05) and high-
amplitude(ΔF/F0peak=0.78(0.24;p<0.05)Ca2þpeaks
were respectively detected after the US (Figure 7a) and
the US þ BTNP stimulations (Figure 7b), suggesting
that the mechano-sensitive cation channels are not at
the base of the mechanism describing the effects
observed during the stimulations.
Finally, in order to investigate whether the high-
amplitude Ca2þtransients observed in the US þ BTNP
stimulation areindeedduetoapiezoelectric effect, we
performed stimulation in the presence of BTNPs char-
acterizedbyacubiccrystallineconfiguration(Figure7c),
and thus not piezoelectric.25Interestingly, we observed
that in this case the US þ BTNP stimulation was able
to induce only low-amplitude Ca2þtransients (ΔF/F0
peak = 0.12 ( 0.01), not significantly different to those
observed after the plain US stimulation (0.15 ( 0.04;
p<0.05),andsignificantlylowercomparedtothehigh-
amplitude peaks induced by the US stimulation in the
presence of BTNPs characterized by a tetragonal crys-
talline structure (p < 0.05).
A representative Ca2þimaging time-lapse frame is
reported in the inlet of each graph of Figure 7, while
more time-lapse images are provided in Figure S6 as
Supporting Information. The XRD analysis of BTNPs
characterized by a cubic crystalline structure is also
reported in Supporting Information, as Figure S7.
DISCUSSION
Several in vitro studies have focused on the be-
havior of different cell types interacting with piezo-
electric substrates/scaffolds. Detailed analysis of
Figure 6. Calcium sources involved during US and US þ BTNP stimulations (0.8 W/cm2). Representative ΔF/F0traces relative
to calcium imaging time-lapses of SH-SY5Y-derived neurons in calcium-free conditions stimulated by US (a) and US þ BTNPs
(b)showinbothcaselow-amplitudecalciumtransients.ObservedtransientswerecompletelyhinderedbydepletingtheCa2þ
fluxfromtheendoplasmicreticulumwiththapsigarginbeforeboththeUS(c)andUSþBTNP(d)stimulations.Arrowsindicate
themomentwhenthe5-sUSpulsewasinitiated;intheinletofeachgrapharepresentativecalciumimagingtime-lapseframe
is reported (at t = 50).
ARTICLE
Page 8
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7685
cytocompatibility and adhesion of human skin
fibroblasts on electruspun poly(vinylidene fluoride-
trifluoroethylene) (PVDF-TrFE) scaffolds have been for
examplecarriedoutbytheArinzehgroup.36Inanother
work, Martins et al. investigated the biological effects
of piezoelectric PVDF fiber polarization and alignment
on the myoblast cell adhesion and morphology for
muscles tissue engineering purposes.37Concerning
neuron-piezoelectric substrate interactions, an inter-
esting work of Lee et al. demonstrated the potential of
micron-sized aligned PVDF-TrFE substrates in promot-
ing and guiding the neurite outgrowth of dorsal root
ganglionneurons.38Theseresultsareparticularlyinter-
esting in view of exploiting piezoelectric substrates for
promoting the neural tissue repair. Furthermore, in
another work of the same group, the neural differen-
tiation of human neural stem/progenitor cells (hNS/
PCs) resulted significantly enhanced in terms of β-III
tubulin positive cells and average neurite length when
carried out on annealed (and thus characterized by
improved piezoelectric properties) vs as-cast PVDF-TrFE
scaffolds.39Obtained results were observed without
making use of any mechanical stimulation, and authors
hypothesized that fiber piezoelectricity was likely trig-
gered by the forces exerted by the cells throughout
adhesion and migration.40In other approaches, instead,
piezoelectric PVDF scaffolds were able to generate an
alternatingelectricfieldontheirsurfaceswhenmechani-
cally deformed by a custom-made vibration base.
A neurite length increase of rat spinal cord neurons
was reported on stimulated piezoelectric PVDF sub-
stratescomparedtononpiezoelectricstimulatedcontrol
scaffolds.24Alternatively, a dedicated device allowed to
apply specific tension to the substrate by regulating the
vacuum pressure in order to piezoelectrically stimulate
fibroblasts cultured on polyurethane/PVDF scaffolds.41
Finally, Inaoka et al. demonstrated asacoustic vibrations
were also able to excite a piezoelectric membrane and,
consequently, to generate electrical signals. The ampli-
fied signals were than transferred to the cochlea of
deafened guinea pigs, so artificially mimicking the func-
tions of the cochlear epithelium.42
In this work we successfully exploited for the first
timepiezoelectricBTNPs,characterizedbyatetragonal
crystallineconfiguration,foranUS-drivenpiezoelectric
stimulation of SH-SY5Y-derived neurons. Primarily, we
observed as BTNPs functionalized with gum Arabic are
associated with the plasma membrane of cells after
24 h of incubation. The observed low internalization of
BTNPs is likely due to their negative external charge,
which is known to limit the nanoparticle up-take.43
However, the localization of the BTNPs at the neural
plasma membrane, which is enriched of voltage-gated
channels and it is electrically excitable, is optimal with
the perspective of a piezoelectric stimulation.44The
US þ BTNP stimulation was able to activate high-
amplitude Ca2þtransients, while the simple US stimu-
lation, without BTNPs, induce low-amplitude Ca2þ
Figure 7. Calcium transients induced by US þ BTNP stimulation (0.8 W/cm2) are mediated by the piezoelectricity of the
nanoparticles.RepresentativeΔF/F0tracesrelativetocalciumimagingtime-lapsesofSH-SY5Y-derivedneuronsstimulatedby
US(a)andUSþBTNPs(b)inthepresenceofgentamicin,ablockerofmechano-sensitivecationchannelswhichdoesnotaffect
the voltage-gated Ca2þcurrents. In (c) the Ca2þtime course of neurons stimulated by US and nonpiezoelectric BTNPs,
characterized by a cubic crystalline configuration. Arrows indicate the moment when the 5-s US pulse was initiated; in the
inlet of each graph a representative calcium imaging time-lapse frame is reported (at t = 50).
ARTICLE
Page 9
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7686
transients. The nonstereotyped amplitude and the
duration (in the order of minute) of the transients
observedaftertheUSþBTNPstimulationsuggest that
these transient are Ca2þwaves.45These phenomena
are known to be dependent on extracellular Ca2þ
and mediated by voltage-gated Naþand Ca2þchan-
nels.46,47Furthermore, it is well-known from the litera-
ture as the Ca2þwaves, both spontaneous and stimuli-
induced, intercellularly propagate through adjacent
neurons thanks to gap junctions.46Interestingly, we
found as some of the observed high-amplitude cal-
cium transients do not occur simultaneously to the
US þ BTNP stimulation: a possible explanation of this
phenomenon is related to the possibility that other
activatedneuronspropagatetheexcitationtoadjacent
neurons. Calcium waves are known to play a key role
in promoting the maturation of the neural network,
especially by regulating the neurite outgrowth:45at
this regard, it is worth to mention as we previously
demonstrated how the US-driven stimulation of piezo-
electric BNNTs was able to significantly promote the
neurite elongation of PC12 neural-like cells, compared
to the US control stimulation without BNNTs. In that
work, it was hypothesized that Ca2þinflux was in-
volved in the increase of the neurite outgrowth, since
this was sensible to the nonspecific Ca2þinflux block-
ers. The findings presented in the current work are
consistent with that hypothesis, having observed how
anUS-drivenpiezoelectricstimulationisabletoinduce
calcium influx.
The high-amplitude Ca2þtransients recorded fol-
lowing the US þ BTNP stimulation are sensitive to TTX
and Cd2þtreatment, suggesting that both Naþand
Ca2þvoltage-gated channels are involved. In particu-
lar, it is possible to argue that the opening of voltage-
gated Naþchannels induces a depolarization of the
plasma membrane, thus activates the voltage-gated
Ca2þchannels, and, finally, increases the cytoplasmic
Ca2þconcentration:48indeed,theTTXþUSþBTNPex-
periments revealed as the voltage-gated Ca2þchan-
nelswerenotsufficient,alone,toevoke high-amplitude
Ca2þtransients. The opening of these voltage-gated
channels in response to the US þ BTNP stimulus is
supposed to induce, as final outcome, a depolarization
of the neuronal plasma membrane.49
Since the voltage-gated channels are expressed on
the plasma membrane, the extracellular Ca2þinflux is
likely involved in the generation of the observed high-
amplitude Ca2þtransients. The US þ BTNP stimulation
in Ca2þ-free conditions reveals a significant decrease
of the Ca2þtransient amplitude, confirming the role of
the extracellular Ca2þinflux. However, in these condi-
tions, it is possible to detect low-amplitude transients,
similar to those measured in response to the US
stimulation in standard conditions (with 2 mM of
extracellularCa2þ),suggestingthatasmallcomponent
of the cytoplasmic Ca2þincrease is due to a release
from intracellular Ca2þstores. Moreover, it is also
possible to detect similar low-amplitude Ca2þtransi-
ents in response to the simple US stimulation in
Ca2þ-free conditions. These results suggest that the
US stimulation without BTNPs is able to induce low-
amplitude Ca2þtransients triggering a release from
intracellularCa2þstores.BydepletingtheCa2þfromER
and in extracellular Ca2þ-free conditions, it is possible
to completely eliminate the Ca2þtransients in re-
sponse to both US and to US þ BTNPs. This finding
confirms the contribution of the ER in the US-depen-
dent increase of the cytoplasmic Ca2þconcentration.
In this context, it is well-known from the literature
thatUS stimulations higher than0.5W/cm2caninduce
a temperature increase,28,17,29which results into Ca2þ
transients upon Ca2þrelease from ER.30Particularly,
the temperature-dependent Ca2þrelease from the ER
islikely evoked byanimbalance betweenanincreased
SERCA pump activity30,31,50and a decreased open
probability of IP3R.30,31,51,52Interestingly, we noticed
as the US stimulation was able to increase the ER
temperature independently by the presence of BTNPs,
suggesting a possible role of the temperature in the
US-induced Ca2þrelease from ER. Since there is not a
significantly different temperature increment follow-
ing the US and the US þ BTNP stimulations, it is pos-
sible to confirm that the high-amplitude Ca2þtransi-
ents evoked by US þ BTNPs are not caused by a
temperature increment, but instead induced by the
voltage-gated channel opening.
Inordertoassesswhethertheobservedphenomena
were actually ascribable to the piezoelectric properties
of the nanoparticles, that act as nanotransducers by
inducing the opening of the voltage-gated channels
in response to the US stimulation, we performed
analogue experiments with US þ BTNPs characterized
by a cubic crystalline configuration, thus not piezo-
electric.Indeed,thisstimulationwasnotabletoinduce
high-amplitude Ca2þtransients, confirming that the
piezoelectricity of the tetragonal BTNPs is a necessary
requirement for eliciting the observed high-amplitude
Ca2þtransients.
This hypothesis was corroborated by a simple elec-
troelasticmodelofaBTNPsubjectedtoUSstimulation.
In particular, the model provides the following expres-
sion for the maximum electric potential increment jR
generated by the US on the BTNP surface (r = R) with
respect to the stress-free condition:
jR? ?R(serrþ2erθ)
sεrr
pUS
sγþ2R
??
(1)
In eq 1, R denotes the radius, errand erθthe piezo-
electric coefficients, and εrrthe dielectric constant of
the BTNP. Moreover, R, γ and s are known expressions
also depending on the elastic properties of the BTNP
(namely the Young modulus and the Poisson ratio).
Finally, pUSdenotes the maximum pressure associated
ARTICLE
Page 10
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7687
withtheUSwave.Themodelderivationisfullydetailed
in the Section S1 of Supporting Information, and
relevant parameter values are discussed/referenced
therein. On the basis of eq 1, when stimulating with
0.1 W/cm2the maximum voltage is around 0.07 mV,
yet it rises to 0.19 mV when operating with 0.8 W/cm2.
In light of the BTNP clusters on the cell mem-
brane (clusters of about 10 BTNPs in average have
been quantified from confocal images), when using
0.8 W/cm2the induced voltage of a few mV can locally
affect channel open probability,49by superposition.
Furthermore, this induced voltage can virtually redis-
tribute the charges of the bivalent ions in correspon-
denceoftheexternalsurfaceoftheplasmamembrane
and, consequently, enhance the voltage sensitivity of
the voltage-gated channels through a shift of the
channel activation curves.53Differently, when using
0.1 W/cm2, it is more difficult for the voltage to affect
channel activation, even where the BTNPs cluster. The
above model predictions are fully compatible with the
experimental observations, and they quantitatively
foster the hypothesis of BTNP-mediated, piezoelectric
cell stimulation.
CONCLUSION
Successfully US-driven piezoelectric neural stimula-
tion was performed by exploiting BTNPs, and the
involved ion fluxes at the base of this phenomenon
werefullydescribed.TheUSþBTNPstimulationcanbe
considered a novel tool for a wireless neural stimula-
tion. Future works will be devoted to the functionaliza-
tion of the BTNPs with specific molecules, in order to
target nanoparticles to the membranes of specific
cell types: cell type selectivity will be in fact funda-
mental envisaging in vivo wireless stimulations of
different parts of the brain, and in order to foster
peculiar cellular functions. Obtained results, collec-
tively, open new intriguing perspectives not only in
the field of neural prosthetics, but also in tissue en-
gineering and biorobotics.
METHODS
BTNP Characterization. Barium titanate nanoparticles were
purchased by Nanostructured & Amorphous Materials, Inc.,
Houston, TX (1144DY). Details of sample purity and composi-
tion, as provided by the supplier, include the following: BaO/
TiO20.999?1.001, purity 99.9%; APS 300 nm; SSA 3.5?3.7 m2/g.
X-ray diffraction (XRD) patterns were recorded using an X-ray
powder diffractometer (Kristalloflex 810, Siemens) using Cu KR
radiation (λ = 1.5406 A) at a scanning rate of 0.016? s?1with 2θ
ranging in 10??80? at a temperature of 25 ?C.
For use in biological experiments, BTNPs were dispersed in
aqueous environment through a noncovalent wrapping with
gum Arabic (G9752 from Sigma-Aldrich). Briefly, 10 mg of
nanoparticles and 10 mg of gum Arabic were mixed in 10 mL
of phosphate buffered saline (PBS) solution. The samples were
sonicated for 12 h with a Bransonic sonicator 2510, by using an
output power of 20 W. The final product is a stable 1 mg/mL
nanoparticle dispersion, that was appropriately diluted in cell
culture medium for biological experiments. Obtained disper-
sion was characterized through scanning electron microscopy
(SEM, Helios NanoLab 600i FIB/SEM, FEI) and transmission
electron microscopy (TEM, Zeiss 902). Moreover, particle size
distribution and Z-potential were analyzed with a Nano Z-Sizer
90 (Malvern Instrument), both in water and in experimental
conditions (50 μg/mL of BTNPs in artificial cerebrospinal fluid,
see in the following for details).
For a control experiment, analogous nanoparticles but
with cubic crystal structure (1143DY, from Nanostructured &
Amorphous Materials) were used following the same prepara-
tion procedures.
CellCulture, Differentiation
neuroblastoma-derived cells (SH-SY5Y, ATCC CRL-2266) were
cultured in DMEM/F12 with 10% fetal bovine serum (FBS,
Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin on
35 mm diameter μ-dishes (Ibidi) at a density of 20000 cell/cm2,
and subsequently differentiated toward neurons in DMEM with
1% FBS, 10 μM all-trans-retinoic acid, 100 U/mL penicillin,
and 100 μg/mL streptomycin. After 4 days of differentiation,
SH-SY5Y-derived neurons were treated for 24 h with BTNPs at
the final concentration of 50 μg/mL in the differentiation
medium. Thisnanoparticleconcentration waspreviously tested
on the SH-SY5Y cell line and was demonstrated to be not toxic
by performing several independent biocompatibility investiga-
tions in terms of proliferation, metabolic activity, apoptosis
andBTNP Treatment. Human
detection and reactive oxygen species detection.54Control
neurons were instead treated for 24 h with the differentia-
tion medium and vehicle of the nanoparticles (50 μg/mL gum
Arabic).
Furthercellviabilityevaluationsinthepresentexperimental
conditionswereperformedwithWST-1assay((2-(4-iodophenyl)-
3-(4-nitophenyl)-5-(2,4disulfophenyl)-2H-tetrazoilium monosodium
salt, provided in a premix electrocoupling solution, BioVision)
and propidium iodide staining, in order to respectively assess
metabolicactivityandmembraneintegrityafterthestimulation
procedures.Moreindetails,culturesundergonethestimulation
protocol (described in the next paragraph) were incubated for
further24h,thereafterculturemediumwasreplacedwitha1:11
premix:medium solution, and incubation performed for further
2 h. Finally, the absorbance was read at 450 nm with a micro-
plate reader (Victor3, PerkinElmer). Membrane integrity evalua-
tion following the stimulation procedure was performed by
adding 1 μg/mL of propidium iodide (PI, Molecular Probes)
during the application of the ultrasounds, and assessing the
number of PI-positive cells over the total number of cells
through fluorescence microscopy (TE2000U, Nikon).
The analysis of the nanoparticle localization was performed
by confocal fluorescence microscopy (FluoView FV1000,
PLAPON 60XO, NA1.42, Olympus). Neuronal plasma mem-
branes and nuclei were stained with CellMask Green Plasma
Membrane Stain (1:1000, Invitrogen) and Hoechst 33342
(1 μg/mL, Invitrogen), respectively. The ER staining in live cells
was performed by using ER tracker Green (500 nM, Invitrogen)
and ER thermo yellow32(300 nM). BTNPs, CellMask Green,
Hoechst 33342, ER tracker Green and ER thermo yellow were
excited by 633, 488, 405, 488, and 543 nm lasers, and the emis-
sion lights were collected at 645?745, 500?555, 425?525,
500?555 and 555?655 nm, respectively.
Ca2þ, Naþand Temperature Imaging during US Stimulation. Before
the stimulation experiments, Fluo-4 AM (1 μM, Invitrogen),
CoroNa Green AM (1 μM, Invitrogen) or the ER thermo yellow32
(300 nM) were incubated in serum-free DMEM for 30 min at
37 ?C. After the reagent incubation, samples were washed,
supplied with artificial cerebrospinal fluid (aCSF, composition
inmM:NaCl140,KCl5,CaCl22,MgCl22,HEPES10, D-glucose10;
pH7.4)andpositionedonaninvertedfluorescencemicroscope.
Fluo-4 and ER thermo yellow were imaged with a microscope
IX81 (Olympus) equipped with an objective UPLFLN 40XO, NA
1.3, and a cooled CCD camera (Cool SNAP HQ2, Photometrics)
ARTICLE
Page 11
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7688
by using 460?480HQ and 535?555HQ as excitation filters,
DM485 and DM565HQ as dichroic mirrors, and 495?540HQ
and 570?625HQ as emission filters, respectively (all from
Olympus). CoroNa Green was imaged with a microscope IX83
(Olympus) equipped with an objective UPLFLN 40XO, NA 1.3,
and an electron multiplying charge-coupled device camera
(iXon3, Andor Technology) by using BP470?495, DM505, and
BP510?550 as an excitation filter, a dichroic mirror and an
emission filter, respectively (all from Olympus).
Concerning the temperature imaging experiments, calibra-
tion was performed by using a near-infrared laser (1064 nm) as
previously described,32and a sensitivity of ?2.0%/?C relative to
room temperature (24 ?C) was measured (please refer to
Supporting Information, Figure S5, for the calibration curve).
Stimulation was carried out after waiting 20 min for the
stabilization of the cell conditions after the medium change,
and for allowing the full de-esterification of the AM groups.
Ultrasounds were applied with a Sonitron GTS Sonoporation
System (ST-GTS, Nepagene) equipped with a plane wave trans-
ducer module (PW-1.0?6 mm, 6 mm diameter tip, 1 MHz). The
probe was vertically fixed at a distance of 5 mm from the cells,
and the US stimulations were carried out for 5 s at different
intensities (from 0.1 W/cm2to 0.8 W/cm2).
During the stimulation experiments, cadmium chloride
(CdCl2100 μM, Sigma), tetrodotoxin (TTX, 100 nM, Tocris), eth-
ylene glycoltetraaceticacid (EGTA, 5 mM, Dojindo Laboratories),
thapsigargin (2 μM, Sigma) or gentamicin (200 μM, Invitrogen)
were added to theaCSF(Ca2þ-freeaCSF was used in the case of
the EGTA experiments). Concerning the thapsigargin experi-
ments, this reagent was also applied during the dye internaliza-
tionprocess,inordertoallowthecompletereleaseofCa2þfrom
the ER, as previously reported.55
Image and Statistical Analysis. The fluorescence images ac-
quired during the time lapses were analyzed with ImageJ
software (http://rsbweb.nih.gov/ij/). Images were thresholded
in order to define the region of interest (ROI) to analyze;
subsequently,theywereconvertedin ΔF/F0byusing thedivide
and subtract functions of the Math process. After a double
smoothing,theaveragevaluesofthepixelsinsidetheROIswere
measured by using the multimeasure function of the ROI
manager and, finally, plotted on the ΔF/F0graphs. Transient
amplitudes (ΔF/F0 peaks) 3-fold higher than the standard
deviation of the noise were reported in terms of peak average
( standard error.
All the described experiments were carried out in triplicate
(at least the response of 20 cells for all the conditions was
analyzed), the normality of all the data distributions was tested
with the Shapiro normality test and, subsequently, the ANOVA
parametric test followed by Tukey's HSD post-hoc test was
performed in order to compare the different distributions.
Conflict of Interest: The authors declare no competing
financial interest.
Acknowledgment. The authors gratefully thank Mr. Piero
Narducci (Department of Chemical Engineering, University of
Pisa, Pisa, Italy) for XRD technical assistance. This research was
partially supported by the JSPS KAKENHI Grant Number
26107717 (to M.S.), by the JSPS Core-to-Core Program, A.
Advanced Research Networks (to M.S.), and by the Italian
Ministry of Health Grant Number RF-2011-02350464 (to G.C.).
Supporting Information Available: Section S1 contains an
electroelastic modelofthevoltage generatedbyapiezoelectric
BTNP subjected to ultrasounds. Figure S1 reports cell viability
data following treatment with BTNPs and US. Figures S2, S3, S4
and S6 report time-lapse frames of the ΔF/F0traces reported in
Figures 4, 5, 6 and 7, respectively. Figure S5 shows the ER
temperatureimagingperformedduringtheUS(0.8W/cm2)and
BTNP þ US (0.8 W/cm2) stimulations. Figure S7 reports the XRD
analysis of BTNPs characterized by a cubic crystalline structure.
Video S1 shows the confocal z-stack 3D rendering of a cluster
of cells (plasma membrane in green, nuclei in blue) and BTNPs
(inred)associatedwiththeirplasmamembrane.VideoS2shows
a Ca2þimaging time-lapse course (18X accelerated) performed
on cultures stimulated with US (0.8 W/cm2) in the presence
of BTNPs. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/
acsnano.5b03162.
REFERENCES AND NOTES
1. Chang,J.-Y.BrainStimulationforNeurologicalandPsychiatric
Disorders, Current Status and Future Direction. J. Pharma-
col. Exp. Ther. 2004, 309, 1–7.
2. Deisseroth, K. Optogenetics. Nat. Methods 2011, 8, 26–29.
3. Gradinaru, V.;Mogri, M.;Thompson, K. R.;Henderson, J. M.;
Deisseroth, K. Optical Deconstruction of Parkinsonian
Neural Circuitry. Science 2009, 324, 354–359.
4. Tye, K. M.; Prakash, R.; Kim, S.-Y.; Fenno, L. E.; Grosenick, L.;
Zarabi, H.; Thompson, K. R.; Gradinaru, V.; Ramakrishnan,
C.; Deisseroth, K. Amygdala Circuitry Mediating Reversible
and Bidirectional Control of Anxiety. Nature 2011, 471,
358–362.
5. Witten, I. B.; Lin, S.-C.; Brodsky, M.; Prakash, R.; Diester, I.;
Anikeeva,P.;Gradinaru,V.;Ramakrishnan,C.;Deisseroth,K.
CholinergicInterneuronsControlLocalCircuitActivityand
Cocaine Conditioning. Science 2010, 330, 1677–1681.
6. Chaudhury, D.; Walsh, J. J.; Friedman, A. K.; Juarez, B.; Ku,
S. M.; Koo, J. W.; Ferguson, D.; Tsai, H.-C.; Pomeranz, L.;
Christoffel, D. J.; et al. Rapid Regulation of Depression-
Related Behaviours by Control of Midbrain Dopamine
Neurons. Nature 2013, 493, 532–536.
7. Knöpfel,T.;Lin,M.Z.;Levskaya,A.;Tian,L.;Lin,J.Y.;Boyden,
E. S. Toward the Second Generation of Optogenetic Tools.
J. Neurosci. 2010, 30, 14998–15004.
8. Diester, I.; Kaufman, M. T.; Mogri, M.; Pashaie, R.; Goo, W.;
Yizhar, O.; Ramakrishnan, C.; Deisseroth, K.; Shenoy, K. V.
An Optogenetic Toolbox Designed for Primates. Nat.
Neurosci. 2011, 14, 387–397.
9. Stanley, S. A.; Gagner, J. E.; Damanpour, S.; Yoshida, M.;
Dordick, J. S.; Friedman, J. M. Radio-Wave Heating of Iron
OxideNanoparticlesCanRegulatePlasmaGlucoseinMice.
Science 2012, 336, 604–608.
10. Perlmutter, J. S.; Mink, J. W. Deep Brain Stimulation. Annu.
Rev. Neurosci. 2006, 29, 229–257.
11. Brunoni, A. R.; Nitsche, M. A.; Bolognini, N.; Bikson, M.;
Wagner, T.; Merabet, L.; Edwards, D. J.; Valero-Cabre, A.;
Rotenberg, A.; Pascual-Leone, A.; et al. Clinical Research
with Transcranial Direct Current Stimulation (tDCS): Chal-
lengesandFutureDirections.BrainStimulat.2012,5,175–195.
12. Stagg, C. J.; Nitsche, M. A. Physiological Basis of Transcranial
Direct Current Stimulation. Neuroscientist 2011, 17, 37–53.
13. Hallett, M. Transcranial Magnetic Stimulation and the
Human Brain. Nature 2000, 406, 147–150.
14. Tufail, Y.; Matyushov, A.; Baldwin, N.; Tauchmann, M. L.;
Georges, J.; Yoshihiro, A.; Tillery, S. I. H.; Tyler, W. J. Tran-
scranial Pulsed Ultrasound Stimulates Intact Brain Circuits.
Neuron 2010, 66, 681–694.
15. Li, J.; Fok, L.; Yin, X.; Bartal, G.; Zhang, X. Experimental
Demonstration of an Acoustic Magnifying Hyperlens. Nat.
Mater. 2009, 8, 931–934.
16. Zhang, S.; Yin, L.; Fang, N. Focusing Ultrasound with an
Acoustic Metamaterial Network. Phys. Rev. Lett. 2009, 102,
194301.
17. Tufail, Y.; Yoshihiro, A.; Pati, S.; Li, M. M.; Tyler, W. J.
Ultrasonic Neuromodulation by Brain Stimulation with
Transcranial Ultrasound. Nat. Protoc. 2011, 6, 1453–1470.
18. Wang, X.; Song, J.; Liu, J.; Wang, Z. L. Direct-Current Nano-
generator Driven by Ultrasonic Waves. Science 2007, 316,
102–105.
19. Qin, Y.; Wang, X.; Wang, Z. L. Microfibre?nanowire
Hybrid Structure for Energy Scavenging. Nature 2008,
451, 809–813.
20. Wang, X.; Liu, J.; Song, J.; Wang, Z. L. Integrated Nanogen-
erators in Biofluid. Nano Lett. 2007, 7, 2475–2479.
21. Kim, K.-H.; Kumar, B.; Lee, K. Y.; Park, H.-K.; Lee, J.-H.;
Lee, H. H.; Jun, H.; Lee, D.; Kim, S.-W. Piezoelectric Two-
DimensionalNanosheets/anionicLayerHeterojunctionfor
Efficient Direct Current Power Generation. Sci. Rep. 2013,
10.1038/srep02017.
22. Zhao, Y.; Liao, Q.; Zhang, G.; Zhang, Z.; Liang, Q.; Liao,
X.; Zhang, Y. High Output Piezoelectric Nanocomposite
ARTICLE
Page 12
MARINO ET AL.
VOL. 9
’ NO. 7
’ 7678–7689
’ 2015
www.acsnano.org
7689
Generators Composed of Oriented BaTiO3 NPs@PVDF.
Nano Energy 2015, 11, 719–727.
23. Ciofani, G.; Danti, S.; D'Alessandro, D.; Ricotti, L.; Moscato,
S.;Bertoni,G.;Falqui,A.;Berrettini,S.;Petrini,M.;Mattoli,V.;
etal.EnhancementofNeuriteOutgrowthinNeuronal-Like
Cells Following Boron Nitride Nanotube-Mediated Stimu-
lation. ACS Nano 2010, 4, 6267–6277.
24. Royo-Gascon, N.; Wininger, M.; Scheinbeim, J. I.; Firestein,
B. L.; Craelius, W. Piezoelectric Substrates Promote Neurite
Growth in Rat Spinal Cord Neurons. Ann. Biomed. Eng.
2013, 41, 112–122.
25. Tao, J.; Ma, J.; Wang, Y.; Zhu, X.; Liu, J.; Jiang, X.; Lin, B.; Ren,
Y. Synthesis of Barium Titanate Nanoparticles via a Novel
Electrochemical Route.Mater.Res.Bull.2008,43,639–644.
26. Lee, C. H.; Ruben, P. C. Interaction between Voltage-Gated
Sodium Channels and the Neurotoxin, Tetrodotoxin.
Channels 2008, 2, 407–412.
27. Lacinová, L. Voltage-Dependent Calcium Channels. Gen.
Physiol. Biophys. 2005, 24 (Suppl 1), 1–78.
28. Ter Haar, G. Therapeutic Applications of Ultrasound. Prog.
Biophys. Mol. Biol. 2007, 93, 111–129.
29. O'Brien, W. D. Ultrasound;biophysics Mechanisms. Prog.
Biophys. Mol. Biol. 2007, 93, 212–255.
30. Itoh,H.;Oyama,K.;Suzuki,M.;Ishiwata,S.MicroscopicHeat
Pulse-Induced Calcium Dynamics in Single WI-38 Fibro-
blasts. Biophysics 2014, 10, 109–119.
31. Tseeb, V.; Suzuki, M.; Oyama, K.; Iwai, K.; Ishiwata, S. Highly
Thermosensitive Ca2þ Dynamics in a HeLa Cell through
IP3 Receptors. HFSP J. 2009, 3, 117–123.
32. Arai, S.; Lee, S.-C.; Zhai, D.; Suzuki, M.; Chang, Y. T. A
Molecular Fluorescent Probe for Targeted Visualization
of Temperature at the Endoplasmic Reticulum. Sci. Rep.
2014, 4, 6701.
33. Calabrese, B.; Manzi, S.; Pellegrini, M.; Pellegrino, M.
Stretch-Activated Cation Channels of Leech Neurons:
Characterization and Role in Neurite Outgrowth. Eur. J.
Neurosci. 1999, 11, 2275–2284.
34. Barsanti, C.; Pellegrini, M.; Ricci, D.; Pellegrino, M. Effects of
Intracellular pH and Ca2þon the Activity of Stretch-
Sensitive Cation Channels in Leech Neurons. Pfluegers
Arch. 2006, 452, 435–443.
35. Jacques-Fricke, B. T.; Seow, Y.; Gottlieb, P. A.; Sachs, F.;
Gomez, T. M. Ca2þInflux through Mechanosensitive
Channels Inhibits Neurite Outgrowth in Opposition to
Other Influx Pathways and Release from Intracellular
Stores. J. Neurosci. 2006, 26, 5656–5664.
36. Weber, N.; Lee, Y.-S.; Shanmugasundaram, S.; Jaffe, M.;
Arinzeh, T. L. Characterization and in Vitro Cytocompatibility
ofPiezoelectricElectrospunScaffolds.ActaBiomater.2010,6,
3550–3556.
37. Martins, P. M.; Ribeiro, S.; Ribeiro, C.; Sencadas, V.; Gomes,
A. C.; Gama, F. M.; Lanceros-Méndez, S. Effect of Poling
State and Morphology of Piezoelectric Poly(vinylidene
Fluoride) Membranes for Skeletal Muscle Tissue Engineer-
ing. RSC Adv. 2013, 3, 17938–17944.
38. Lee, Y.-S.; Collins, G.; Arinzeh, T. L. Neurite Extension of
Primary Neurons on Electrospun Piezoelectric Scaffolds.
Acta Biomater. 2011, 7, 3877–3886.
39. Lee, Y.-S.; Arinzeh, T. L. The Influence of Piezoelectric
Scaffolds on Neural Differentiation of Human Neural Stem/
progenitor Cells. Tissue Eng., Part A 2012, 18, 2063–2072.
40. Wang, N.; Toli? c-Nørrelykke, I. M.; Chen, J.; Mijailovich, S. M.;
Butler, J. P.; Fredberg, J. J.; Stamenovi? c, D.; Cell Prestress, I.
Stiffness and Prestress Are Closely Associated in Adherent
Contractile Cells. Am. J. Physiol. Cell Physiol. 2002, 282,
C606–C616.
41. Guo,H.-F.;Li,Z.-S.;Dong,S.-W.;Chen,W.-J.;Deng,L.;Wang,
Y.-F.; Ying, D.-J. Piezoelectric PU/PVDF Electrospun Scaf-
folds for Wound Healing Applications. Colloids Surf., B
2012, 96, 29–36.
42. Inaoka,T.;Shintaku,H.;Nakagawa, T.;Kawano,S.;Ogita,H.;
Sakamoto, T.; Hamanishi, S.; Wada, H.; Ito, J. Piezoelectric
Materials Mimic the Function of the Cochlear Sensory
Epithelium. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 18390–
18395.
43. Fröhlich, E. The Role of Surface Charge in Cellular Uptake
andCytotoxicityofMedicalNanoparticles.Int.J.Nanomed.
2012, 7, 5577–5591.
44. Armstrong, C. M.; Hille, B. Voltage-Gated Ion Channels and
Electrical Excitability. Neuron 1998, 20, 371–380.
45. Rosenberg, S. S.; Spitzer, N. C. Calcium Signaling in Neuronal
Development. Cold Spring Harbor Perspect. Biol. 2011, 3,
a004259.
46. Charles, A. C.; Kodali, S. K.; Tyndale, R. F. Intercellular
Calcium Waves in Neurons. Mol. Cell. Neurosci. 1996, 7,
337–353.
47. Gu, X.; Olson, E. C.; Spitzer, N. C. Spontaneous Neuronal
Calcium Spikes and Waves during Early Differentiation.
J. Neurosci. 1994, 14, 6325–6335.
48. Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Calcium
Signalling: Dynamics, Homeostasis and Remodelling. Nat.
Rev. Mol. Cell Biol. 2003, 4, 517–529.
49. Catterall, W. A. Structure and Function of Voltage-Gated
Ion Channels. Annu. Rev. Biochem. 1995, 64, 493–531.
50. Dode, L.; Van Baelen, K.; Wuytack, F.; Dean, W. L. Low
Temperature Molecular Adaptation of the Skeletal Muscle
Sarco(endo)plasmic Reticulum Ca2þ-ATPase 1 (SERCA 1)
intheWoodFrog(RanaSylvatica).J.Biol.Chem.2001,276,
3911–3919.
51. Stavermann, M.; Buddrus, K.; St John, J. A.; Ekberg, J. A. K.;
Nilius, B.; Deitmer, J. W.; Lohr, C. Temperature-Dependent
Calcium-Induced Calcium Release via InsP3 Receptors in
Mouse Olfactory Ensheathing Glial Cells. Cell Calcium
2012, 52, 113–123.
52. Dickinson, G. D.; Parker, I. Temperature Dependence of
IP3-Mediated Local and Global Ca2þSignals. Biophys. J.
2013, 104, 386–395.
53. Hille,B.ModifiersofGating;SinauerAssociatesInc.:Sunderland,
MA, 1992; Ionic Channels of Excitable Membranes, Vol. 17.
54. Ciofani, G.; Danti, S.; D'Alessandro, D.; Moscato, S.; Petrini,
M.; Menciassi, A. Barium Titanate Nanoparticles: Highly
Cytocompatible Dispersions in Glycol-Chitosan and Dox-
orubicin Complexes for Cancer Therapy. Nanoscale Res.
Lett. 2010, 5, 1093.
55. Paschen, W.; Doutheil, J.; Gissel, C.; Treiman, M. Depletion
of Neuronal Endoplasmic Reticulum Calcium Stores by
Thapsigargin: Effect on Protein Synthesis. J. Neurochem.
1996, 67, 1735–1743.
ARTICLE
Download full-text 
