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IRBM 35 (2014) 94–99

ANR TECSAN 2010

Monitoring and control of inertial cavitation activity for enhancingultrasound transfection: The SonInCaRe project

C. Inserra a,∗, P. Labelle a, C. Der Loughian a,b, J.-L. Lee c, M. Fouqueray a, J. Ngo a,A. Poizat a, C. Desjouy a, B. Munteanu d, C.-W. Lo c, C. Vanbelle a,

J.-P. Rieu b, W.-S. Chen c,e, J.-C. Béra a

a Inserm, U1032, LabTAU, université Claude-Bernard Lyon 1, 69003 Lyon, Franceb Institut Lumière Matière, UMR5306, université de Lyon 1-CNRS, université de Lyon, 69622 Villeurbanne cedex, France

c Department of physical medicine and rehabilitation, national Taiwan university hospital, national Taiwan university college of medicine,No.7, Zhongshan S. road, Taipei 100, Taiwan

d Université de Lyon, CNRS INSA-Lyon, LaMCoS UMR5259, 69621 Villeurbanne cedex, Francee Division of medical engineering research, national health research institute, 35, Keyan road, Zhunan, Miaoli 350, Taiwan

Received 5 February 2014; received in revised form 6 February 2014; accepted 6 February 2014Available online 19 March 2014

bstract

Sonoporation process, at the origin of ultrasound cell transfection, is ruled by the interaction between cells and cavitating bubbles. Due to thetochastic behavior of acoustic cavitation, there exists a need in ensuring a stable state of cavitation within a medium during cell transfection tonhance transfection efficiency and control mortality. The goal of the SonInCaRe project is thus to define a controlled-cavitation device in ordero monitor, control and stabilize inertial cavitation activity during cell sonication in real-time. This device, based on a feedback loop acting in

eal-time, allows ensuring fixed cavitation conditions during a pulsed sonication. Its application to suspended and adherent cells transfection showsetter reproducibility compared to the fixed acoustic intensity sonication. The regulation device thus provides a better control of cavitation activitynd its bioeffects which are of crucial importance for clinical applications of ultrasound-mediated gene transfection.

2014 Elsevier Masson SAS. All rights reserved.

iageioc

. Introduction

Drug delivery and gene therapy have recently opened a poten-ially new perspective for managing an array of complicatediseases that may have substantial societal dimensions. Amonghe possible applications, the administration of a therapeuticene or drug can help in treating pathologies like monogenic

ereditary diseases (mucoviscidose, Duchenne myopathy. . .),ancers, vascular and metabolic diseases [1]. In addition toonsiderable advances in the identification of specific genes

∗ Corresponding author. Inserm, unit 1032, laboratory of therapeutic applica-ions of ultrasound, 151, cours Albert-Thomas, 69424 Lyon cedex 03, France.

E-mail address: claude.inserra@inserm.fr (C. Inserra).

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http://dx.doi.org/10.1016/j.irbm.2014.02.010959-0318/© 2014 Elsevier Masson SAS. All rights reserved.

nvolved in causing, regulating and curing diseases, new ther-peutic modalities have to be found to efficiently deliver theseenes and treat diseases safely and efficiently. Several strategiesxist for drug or gene delivery into cells. Viral methods consistn using virus to transfer genes into cells, while nonviral meth-ds rest upon the use of either synthetic vectors to penetrate theells or external energetic sources to enhance molecular uptake.ecause of the limitations of viral methods like cytopathy,ytotoxicity and an unpredictable immune response, nonviralethods are considered to be safer than viral-mediated molecule

or a therapeutic use [2]. Common nonviral methods (electro-oration, microinjection or gene gun) present limitations that

ltrasound could remove at the condition to obtain a better bal-nce between transfection efficiency and cell viability [3,4]. Thehenomenon of ultrasound enhancement of molecule uptake,

RBM 35 (2014) 94–99 95

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nown as sonoporation, is based on the temporary increase ofell membrane permeability and its poration allowing moleculeransfer [5]. Ultrasound thus reveals many attributes of an idealransfer system, as it is low-invasive, well-tolerated by theatients, cost-effective and can achieve site-specific transfer ofnergy within the body as ultrasound can be focused [6,7].evertheless, the transfection efficiencies of sonoporation are

ower than those of other methods, and many technical prob-ems arise when applied in-vivo. These limitations are generallyhought to be due to the lack of fundamental understanding ofhe mechanisms underlying sonoporation. Even if the mecha-isms leading to cell poration are still not clearly established, thenteraction between cells, ultrasound and microbubbles appearso be the main candidate through the phenomenon of cavitation8–10]. In the presence of ultrasound, microbubbles can oscil-ate around a resonant diameter and generate shear force andcoustic microstreaming, named as the stable cavitation regime11]. For higher acoustic intensities, rapid bubble expansion isollowed by bubble collapse, named as transient or inertial cav-tation [12], leading to high extreme temperatures and pressureonditions, accompanied by radial shock wave emission or freeadical generation. Recent studies have focused on the role ofransient cavitation as being better correlated to the transfectionfficiency compared to stable cavitation, but the exact mech-nism by which inertially cavitating bubbles alter cell porosityemains poorly understood. Particularly, the higher is the appliedcoustic intensity, the higher are both transfection efficiencynd cell toxicity. Considering the chaotic behavior of cavitationnitiation and leveling, and consequently the unpredictable bio-ogical effects, a good balance between transfection efficiencynd cell toxicity could be achieved by controlling acoustic cav-tation in real-time during cell sonication [13]. The main aimf the SonInCaRe project was thus to design, implement andalidate a level-controlled cavitation system applied to in-vitroransfection of cells, with the perspectives of a reproducible andnder control sonoporation. To achieve this goal, fundamentalesearch for a better understanding of the effects of cavitationn the cell membrane, the design of a new technological deviceor sonoporation, and its application to in-vitro cell transfectionave to be performed. In this study, a review of the main resultsf the project are presented by introducing:

the monitoring and control of inertial cavitation activity inreal-time;

the application of the cavitation-controlled device to sus-pended cell transfection and;

preliminary results on the understanding of bubble-cell inter-action.

Concerning the last point, we designed a sonoporation devicellowing real-time visualization to study adherent cell sono-

oration. To get insights in the sonoporation mechanisms, thenteraction between surfacic cavitating bubbles and supportedilayer membranes (considered as a cell monolayer model) aretudied thanks to this device.

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ig. 1. Examples of spectra obtained from the hydrophone signal, exhibitingtable cavitation or inertial cavitation.

. Stochastic behavior of inertial cavitation and itsontrol

Acoustic cavitation occurs when a liquid medium is subjectedo ultrasound at sufficiently high pressure amplitude [14]. Vari-tions of the pressure in the medium can lead to the nucleationnd excitation of micron-sized bubbles. The activity of cavi-ating bubbles is classified into two regimes: stable or inertialavitation. The stable cavitation is characterized by radial linearr non-linear oscillations of the bubble, which give rise to themission of harmonics and sub-harmonics of the fundamentalxcitation frequency (Fig. 1). Inertial cavitation occurs aboveome threshold pressure level for which bubbles can exhibitarge growth and violent collapse characterized by the emissionf a broadband noise (Fig. 1).

As regards transfection applications, most of the authorsypothesize that transfection rate is intimately linked toltrasound-induced inertial cavitation [15]. In this context, mon-toring in real-time the inertial cavitation activity is of greatmportance. In the present investigations, an acoustic monitorings performed by the use of an hydrophone and through the intro-uction of a cavitation index CI(t) that quantified the broadbandoise generated by microbubbles collapses. The calculation ofhe CI(t) consists in evaluating the spectral broadband signalnhancement during the cavitation activity by taking the meanrithmetic value of the overall frequency dB magnitude of thepectrum [16].

Most studies on ultrasound cavitation are performed at fixedcoustic pressures. The use of this type of operating protocolakes the results difficult to analyse because the cavitation phe-

omenon is characterized by a random behavior that greatlyisturbs the reproducibility, initiation and time stability of cav-tation activity. To illustrate the random behavior of inertialavitation, Fig. 2 shows the mean inertial cavitation index CIbtained when using a fixed applied acoustic intensity during a

onication experiment.

Results present a great discrepancy in the inertial cavitationctivity at a given acoustic intensity, resulting in possibly large

96 C. Inserra et al. / IRBM

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coustic intensity or fixed inertial cavitation activity. Average ± standard devi-tions (n = 3).

ariation of cavitation-induced biological effects. For example,or an acoustic intensity Ia = 0.6 W/cm2, the CI value can varyetween 7 (no inertial activity) and 14 (high inertial activity).hen analysing the temporal evolution of the CI(t) value during

n acoustic pulse (Fig. 3), it appears that the CI value is fluctuat-ng in a large range. It is worth noting that the attained extremaould lead to cell mortality. To overcome this stochastic behav-or, a novel strategy allowing the control in real-time of inertialavitation activity in pulsed sonication mode was consequentlyeveloped [17]. The strategy consists in the monitoring in real-ime of the inertial cavitation activity through the indicator CI(t),nd the modulation of the acoustic intensity applied to the trans-

ucers to reach and maintain a fixed inertial cavitation indexuring the pulse. This is achieved within a Field Programmableate Array (FPGA) core (NI PXIe-7965R card) that synthesizes

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35 (2014) 94–99

he output signal feeding the ultrasound transducers, acquires thecoustic pressure signal and makes all the required operationswindowing, FFT calculation, modulation of the acoustic sig-al amplitude. . .). When using the regulated-cavitation mode,he voltage applied to the transducers is consequently vary-ng according to a proportional-gain relationship, and this isone every regulation loop of nearly 300 �s. Fig. 3b presentshe results of the regulation process of the inertial cavitationndex in real-time for three acoustic pulses during a sonicationxperiment. Compared to the fixed acoustic intensity sonication,he level of inertial cavitation activity is maintained at a constantalue, with a good time stability of the regulation process within

single pulse.

. Ultrasound cell transfection in cavitation-controlledonditions

In order to validate the controlled-cavitation device, the real-ime monitoring and control of inertial cavitation activity ismplemented on a sonoporation system for suspended cell trans-ection [18]. The ultrasound set-up consists in a tank filledith degassed water, containing a 24-well plate located above

1 MHz plane piezoelectric transducer. BNL 1MEA.7R.1 cellschemically-transformed liver cells) are grown in cell mediumontaining 4.5 g/L glucose, 10% fetal bovine serum, a 1% mix-ure of penicillin G, streptomycin and amphotericin B at 37 ◦Cn 5% CO2. Cells are placed in each well of the plate at a densityf 5 104 in 1 mL of cell medium. In each well, 2 mL of cultureedium with 10 �g of luciferase plasmid and 5 �L of SonoVue

ontrast agent are added. To evaluate transfection efficiency of

stimated by a LSRII flow cytometer (Becton Dicksinson, CA,SA). More details on the experimental set-up and analysis areiven in [18].

0 10 20 30 40 50-5

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ied acoustic intensity and (b) fixed inertial cavitation mode. Three consecutive

C. Inserra et al. / IRBM 35 (2014) 94–99 97

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ig. 4. Transfection efficiency of luciferase in BNL cells in fixed intensity modeIa) or fixed cavitation mode (CI). Average ± standard deviations (n = 5).

Fig. 4 presents the results in terms of transfection efficiencyf reported genes when sonicating cells at a fixed acoustic inten-ity of 0.21, 0.31 and 0.41 W/cm2 or at a fixed cavitation indexf 7, 10 and 16. It is worth noting first that luciferase is suc-essfully transfected into cells whatever the irradiation mode.hen applying a fixed acoustic intensity, huge variations in the

ransfection efficiency are noticed, whatever the applied inten-ity. This result highlights the effect of cavitation randomnessn biological phenomenon. Indeed, following in real-time theavitation activity during a fixed intensity sonication revealshat, due to the random behavior of cavitation, cavitation activityaries over a wide range of CI values during a single experimentFig. 2). On the contrary, when using the fixed cavitation mode,ransfection efficiency is more reproducible and monotonouslyepends on the CI value. This correlation indicates, as expected,hat sonoporation phenomena are linked to the inertial cavita-ion activity instead of the applied acoustic intensity, and thatiological effects could be better controlled when using theavitation-controlled device.

. Towards mechanisms underlying cell sonoporation

Usually ultrasound transfection is performed on suspendedells for two main reasons:

the simplicity of the technological ultrasound set-up for son-icating a whole volume and;

better transfection efficiency for suspended cells in compari-son to adherent cells.

In order to get some insight on the fundamental mechanismsnderlying sonoporation, developing a sonoporation device ded-cated to adherent cells is of particular interest as it could allowisualizing bubble-cell interactions, which is difficult for sus-ended cells where interactions are in the whole volume. Inhe following part, we consequently present a new sonoporation

evice adapted to a microscope stage, dedicated to the sonopora-ion of adherent cells, and preliminary results on bubble activityear a substrate are provided.

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ig. 5. Photograph of the sonoporation device dedicated to the sonoporation ofdherent cell monolayer.

.1. Real-time visualization sonoporation device

In order to visualize bubble-cell interaction and in the aim ofetting information on sonoporation process, an ultrasound set-p has been mounted on a microscope stage allowing real-timeisualization of bubble activity atop an adherent cell mono-ayer during a sonication experiment. A photograph of theesigned microscope stage is presented in Fig. 5. At the centerf the stage is located a culture well (Labtek, 2 wells, dimen-ion 20 × 20 mm). On each well, on the two opposite sides arelaced face-to-face two plane piezoelectric transducers (Fer-operm PZ26, frequency 426.5 kHz, dimension 18 × 3 mm).his configuration allows letting free the vertical axis (perpen-icularly to the stage) for the optical path. The transducers,lectrically matched to 50 �, generate a continuous or pulsedltrasound wave provided by a function generator amplifiedy a power amplifier. The electrical power delivered by theenerator is ranged between 0 and 8 W, corresponding to max-mum acoustic intensity of 6.8 W/cm2. A needle hydrophoneOnda HNR-0500) placed on another well side allows lis-ening the cavitation activity in the exposure medium. Theydrophone signal is then digitized through the FPGA core thatllows regulating the inertial cavitation activity, as described inection 2.

.2. Cavitation on a lipid bilayer membrane

Before assessing the transfection efficiency of adherent cellsith the designed device, the potential of generating cavitationubbles in the vicinity of the well’s bottom is first evaluated,s the initiation and location of bubbles near a wall is not triv-al. Indeed, while radial dynamics [19] and space motion [20]f bubbles in fluid volume have been extensively studied, fewtudies concern the dynamics or behavior of bubbles in the vicin-ty of a wall, at least experimentally [21,22]. To get insights of

he surfacic cavitation at the bottom of the well, a supportedipid bilayer membrane has been deposited at the bottom of theell as an indicator of bubble presence and interaction with this

98 C. Inserra et al. / IRBM 35 (2014) 94–99

Fig. 6. Confocal observation of cavitating bubbles near the culture well bottom and their effect on lipid bilayer membrane integrity. First line, transmission imagess d by

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howing bubbles; second line, fluorescence images showing the damages causen the bottom right is due to photobleaching during a previous scan).

all. DPPC or DLPC bilayers (gel and fluid phases at roomemperature respectively) with 2% of the fluorophore NBD-PCall Avanti Polar Lipids) were deposited at the bottom of the welly the vesicle fusion method. The well is placed in the micro-cope stage described in section 4.1 and located under a confocalicroscope (Leica TCS SP5 II). Observations are performed

oth in transmission (natural light) for bubble observation anduorescence (488 nm excitation) for lipid observation during aonication. Fig. 6 presents the cavitation activity in the vicin-ty of the lipid bilayer as a function of exposure time. Beforepplying ultrasound (Fig. 6a), no bubbles are present at the bot-om, and the lipid bilayer exhibits a uniform fluorescence (intact

embrane). In the bottom right-hand corner, a dark square onhe membrane is observed and results from a long observationf the lipid bilayer with laser: this zone endured photobleach-ng. When applying ultrasound (Fig. 6b), a bubble cloud appearsn natural light, and circular impacts are observed on the lipidilayer at the same location. The fluorescence increase denoteshe presence of surfacic bubbles which attracted lipids from theilayer, while darker zones reveal lipid detachment from theilayer. This confirms that cavitation bubbles interact at the bot-om of the well with the lipid bilayer. When the bubble cloudpatially extends (Fig. 6c), the same behavior is observed onhe lipid bilayer. Interestingly, in the right-hand corner of thegure, cracks appear on the membrane far from the bubble loca-

ion. These cracks could result from the temporary passing of

ast microbubbles in this zone, not visible in natural light butetting some permanent damages on the membrane: the lipidilayer then could be used as a memory effect marker of theubble presence near the bottom of the well. When the bubble

wsnm

cavitating bubbles to an originally homogeneous supported bilayer (the square

loud translates in the top right-hand corner, the lipid bilayerresents dark zones of lipid detachment at the previous locationf the cavitation cloud. These permanent damages confirm thatnteraction between adherent cells and bubbles could exist at theottom of the well.

.3. Observation of sonoporated cells

Concerning the device validation for adherent cell transfec-ion, HT29 adherent cells (human colorectal adenocarcinomaell line) were plated in the well at 2 106 cells/well, with

mL of RPMI 1640 completed media. Sonoporation experi-ents consisted in the internalization of siRNA coupled with

he fluorochrome AlexaFluor-488, before analysis with a flowytometer for providing viability and sonoporation rate. Resultsonfirmed that adherent cells could be sonoporated at a rateignificantly higher than the endocytosis rate. Moreover, inhe same way that for suspended cell transfection (section 3),onoporation efficiency is more reproducible when using theontrolled-cavitation device than in the fixed applied acous-ic intensity mode. A preliminary experiment on bubble-cellnteractions is implemented during sonication under a confocalicroscope (Zeiss Confocal Laser Scanning Microscopy 780).or visualization, cell membranes have been colored in red usingybrant DID and siRNA-AlexaFluor 488 molecules appear inreen. Before sonication, siRNA molecules are spread in the

hole volume and do not appear inside cells. After sonication, a

canning of the well bottom well revealed that siRNA was inter-alized within cells, as shown in Fig. 7. Interestingly, siRNAolecules in green appear to be internalized in the nucleus of

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ig. 7. 3D view (confocal microscope) of sonoporated cells containing siRNAolecules.

he cell, phenomenon that could be interpreted as stable sono-oration.

. Conclusion

To overcome the random behavior of cavitation phenomenon, novel strategy based on the monitoring and control of inertialavitation activity during pulsed sonication has been developed.he designed device showed its efficiency for ensuring initi-tion, leveling and time stability of inertial cavitation activity.eproducible transfection rate were obtained for both suspendednd adherent cells. Implemented in a real-time microscopy sys-em, it provides insights in the interaction between microbubblesnd adherent cells. This system allows the study of the interac-ion between surfacic bubbles and supported bilayer membranes,ith the assessment of permanent or transient damages inducedy cavitation bubbles on the membrane. Moreover, concerningn-vitro transfection mechanisms, the possibility of visualizingiRNA internalization within cells has been demonstrated.

cknowledgments

This work is supported by the French National Researchgency ANR project “SonInCaRe” no. 2010-TECS-003-01,

nd by the National Science Council of the Republicf China, Grant NSC 99-2320-B-002-005-MY3 and 100-923-B-002-003-MY3, and benefits from the Labex CeLyANR-10-LABX-0060.

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