Anthony Delalande

Patrick Midoux

Chantal Pichon
email: chantal.pichon@cnrs-orleans.fr

Spiros Kotopoulis

Michiel Postema

Investigations of microbubble-cell interactions during the sonoporation process

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Activation of microbubbles (MB) under specific ultrasound (US) beams induces a transient cell membrane permeabilization with a process known as sonoporation [START_REF] Bao | Transfection of a reporter plasmid into cultured cells by sonoporation in vitro[END_REF][START_REF] Miller | Sonoporation of cultured cells in the rotating tube exposure system[END_REF][START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF]. Sonoporation is the transient permeabilization and resealing of a cell membrane with help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules up to 3 MDa. Many studies have demonstrated increased drug and gene delivery of sites under US application, presumably owing to sonoporation. In the field of gene transfer, several studies have reported an improvement of gene delivery by US assisted MB [START_REF] Wang | Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents[END_REF]. Although mechanical disruption with the aid of ultrasound has been attributed to violent side effects of inertial cavitation and microbubble fragmentation, most notably, the increased uptake has also been observed at low acoustic amplitudes, i.e., in acoustic regimes where inertial cavitation and microbubble fragmentation are not to be expected. An US contrast agent microbubble might act as a vehicle to carry a drug or gene load to a perfused region of interest. If the same ultrasound field that has been implicated in the sonoporation process can cause release of the therapeutic load, this load could be delivered into cells. Apart from plainly mixing ultrasound contrast agents with therapeutic agents, several schemes have been proposed to incorporate therapeutic loads to microbubbles. Several studies have been conducted these last years to delineate mechanisms involved in US mediated delivery [START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF][START_REF] Duvshani-Eshet | Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy[END_REF][START_REF] Duvshani-Eshet | The effects of albumin-coated microbubbles in DNA delivery mediated by therapeutic ultrasound[END_REF][START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF]. However, it is still unclear exactly how cells that are subjected to US and MB internalize extracellular compounds, and which cellular responses US and MB evoke. As others, we have shown that sonoporation can also induce an outward transport of small intracellular molecules that likely due to membrane destabilization [START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF]. The permeabilization engenders a transient release of small molecules such as enhanced green fluorescent protein (eGFP) from the cytosol of HeLa cells stably expressing eGFP gene while preserving cell viability. Results obtained in that study, using BR14 Bracco's MB, reinforced the hypothesis of transient pore formation mechanism induced by sonoporation. However, this seems not to be the unique mechanism that is involved in sonoporation. Indeed, the contribution of endocytosis besides pore formation for the uptake of extracellular molecules by sonoporation has been proposed recently [START_REF] Meijering | Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. If so, an improved knowledge on both the extracellular and intracellular fates of MB and the cargo need to be acquired to clearly specify limitations and to further improve the system. The study aimed at acquiring knowledge on microbubble-cell interactions during sonoporation by following the intracellular fate of microbubbles and plasmid DNA (pDNA) as cargo inside cancer cells. Investigations were performed on HeLa cells, human cervix cancer cells that stably express protein markers of intracellular organelles which are fused with enhanced green fluorescent protein (eGFP) as fluorescent tag. We have established these cells to delineate intracellular routing of pDNA delivered by our chemical vectors [START_REF] Pichon | Chemical vectors for gene delivery: uptake and intracellular trafficking[END_REF] and Billiet et al., 2011 under submission). Data shown were obtained with HeLa cells expressing either the small GTPase Rab7-eGFP or the nucleoporin Nup153-eGFP. Rab7 and Nup153 are commonly used as marker of late endosomes and nuclear pore, respectively. Therefore, these fluorescent chimera molecules are useful to delimit easily the late endosomal compartments and the nucleus in live cells. Sonoporation was performed with Micromarker TM bubbles. The pDNA and MB were labelled with fluorescent tags to follow their localization inside cells upon sonoporation.

Material and Methods

Cell culture

HeLa cells (ATCC CCL-2) were immortalized cells derived from the cervix adenocarcinoma of a young American female. HeLa-Rab7-eGFP and HeLa Nup153-eGFP cells are HeLa cells that were stably transfected with plasmid encoding Rab7 and Nup153 genes fused with eGFP gene, respectively. The stable clones used in this study were obtained upon cell sorting by flow cytometry. Cells were cultured in MEM with Earl's salts medium supplemented with 10% v/v heat-inactivated fetal calf serum (FCS), GlutaMAX TM (LifeTechnologies Gibco, Paisley, Renfrewshire, UK), 1% v/v of non-essential amino-acids, penicillin (100 units/ml) and streptomycin (100 µg/ml), at 37°C in a humidified atmosphere containing 5% CO2.

Plasmids

The reporter plasmid DNA used was a homemade plasmid of 7.5 kb that encodes the firefly luciferase gene under control of the strong cytomegalovirus (CMV) promoter. Five consecutive κB motifs that recognize the NFκB transcription factor have been inserted upstream of the promoter [START_REF] Goncalves | An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression[END_REF]. pDNA was labelled with cyanin 3 (Cy3) using the Label IT nucleic acid labelling kit (Mirus, Madison, WI, USA) at 1 : 2 reagent/pDNA weight ratio according to the manufacturer's instructions. The labelled DNA was purified by ethanol precipitation. The labeling density determined by absorbance according to the manufacturer's protocol was 1Cy3 per 80 bp.

Microbubbles and ultrasound set-up

MicroMarker TM MBs were provided from VisualSonics B.V. (Amsterdam, Netherlands). They are composed of perfluorobutan gas encapsulated in a phospholipid shell. Micromarker TM (MM) microbubbles have a mean diameter of 2.3-2.9 µm. MM were prepared a few minutes before experimentation according to the manufacturer's protocol. The experimental set-up used is shown below.

US waves were generated from a 0.5 inches transducer with 1 MHz frequency (IBMF-014, Sofranel, Sartrouville, France). The transducer was driven with an electrical signal generated by an arbitrary waveform generator (Agilent 33220A, Agilent technologies) and amplified with a power amplifier (ADECE, Artannes, France). Pulsed ultrasound (10 kHz) with a duty cycle of 40% was used in this study. The negative peak acoustic pressures investigated were measured in a separate set-up with a calibrated HLG-200 capsule hydrophone (Onda, Dahm International ApS, Denmark) at 3 cm depth, the natural focal distance of the transducer. The hydrophone was positioned inside the insonation cuvette. The attenuation of the cuvette walls was measured separately and found to be negligible at the frequency 1 MHz (less than 10%). We therefore did not expect significant standing waves to be generated inside the cuvette. 10 5 cells were seeded on a center portion of Opticell ® membrane (1 x 1.5 cm) one day prior sonoporation experiments. After washing with Opti-MEM (Invitrogen, Carlsbad, CA) supplemented with 1% FCS, the Opticell ® was placed inside the cuvette and the sonoporation was performed as indicated.

Luciferase assay

The transfection efficiency of pDNA encoding luciferase gene was determined by measuring the luciferase activity in cell lysate. The Relative Light Units (RLU) was measured with a Lumat LB9507 luminometer (Berthold, Wildbarch-Germany) after adding 100 µl of luciferase substrate (1mM luciferin containing ATP) to 60 µl of cell lysates and expressed as RLU per mg of proteins. Proteins content of cell lysates were quantified by bicinchoninic acid assay [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF].

High speed camera and confocal fluorescence microscopy

An overview of the setup used for the fluorescence experiments is shown in Figure 4. A custommade aluminium sonication chamber with internal dimensions of 130 × 170 × 35 mm 3 was locked into to the xy-stage of a 200M inverted confocal microscope (Carl Zeiss AG, Oberkochen, Germa-ny) coupled with a LSM Axiovert 510 scanning device (Carl Zeiss), using an EC Plan-Neofluar 40×/1.30 oil DIC M27 objective (Carl Zeiss AG), with automated z-stack functionality. The peak-negative acoustic pressure was measured at the objective's field of view and corresponded to MI=0.2. An OptiCell ® cell culture chamber (Nunc GmbH & Co. KG, Langenselbold, Germany) was placed on the sonication chamber. On one side of the cell culture chamber was seeded a monolayer HeLa cells (1× 10 6 ). Prior to injection in the OptiCell ® , MicroMarker contrast agent was labelled using a DiD (DilC 18 [START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF], λexc=633 nm and λem= 649-703nm) lipophilic fluorescent probe (Vybrant TM , Molecular probes, Invitrogen, San Diego, CA, USA). A ratio of 1 µl of DiD to 40 µl MicroMarker was homogenized by pipetting and incubating for 5 minutes at room temperature. MBs were injected into the cell culturing chamber before each experiment. The signal consisted of 40 cycles with a centre frequency of 1 MHz and a pulse repetition frequency of 10 kHz, with a maximum MI of 0.15.

Results and discussion

The optimal acoustic parameters that give the maximal gene transfer have been first determined on adherent HeLa cells. Our data indicate that the luciferase gene expression was dependent on the acoustic parameters and the presence of MB. We found that optimal parameters that gave a maximum luciferase gene expression without significant toxicity at 1 MHz of US was 150kPa, 40% duty cycle, 60 sec of exposure time in presence of 10 7 bubbles. As an example, shown in Figure 1 are the levels of luciferase expression as function of acoustic pressure.

A key to success of this technique lies in understanding mechanism governing MB and cell interactions. Real time confocal microscopy investigations were performed on adherent cells using pDNA and MB labeled with Cy3 (red) and DiD (blue) lipophilic tracer, respectively. The first experiments were done on Hela cells with pDNA-tagged Cy3 which was mainly localized as red spots at plasma membrane straightaway following sonoporation. At later time, these spots progressively moved inside cells (not shown). The structures could correspond either to vesicles (endosome) or aggregates like structures upon sonoporation. To distinguish between these 2 possibilities, kinetic experiments were performed in HeLa cells to investigate if pDNA and MB reached some endocytic organelles upon sonoporation. Data presented in figure 2 were obtained with HeLa cells expressing Rab7-eGFP (green) which are localized on the surface of late endosomes, intracellular compartments in which end up internalized molecules before being delivered into lysosomes for degradation. Upon sonoporation, pDNA were localized mainly at the plasma membrane and without colocalization with Rab7-eGFP (figure 2A and2B). Observations made 3 h post-sonoporation before indicated that some of pDNA but not all ended up into late endosomes as demonstrated by yellow staining (merge of green and red spots) indicating their localization in Rab7 positive organelles (figure 2C andD). The presence of punctuate red staining corresponding to pDNA could correspond to pDNA that are either free (present inside the cytosol upon sonoporation) or routed in another intracellular compartments different to late endosomes. MB labelled with DiD were used to follow their fate in HeLa cells expressing Nup153-eGFP that delimit the cell nucleus. The representative analysis shown in Figure 3 was acquired 6h postsonoporation. Strikingly, we found that in addition to pDNA (red) which are observed throughout the cytoplasm as in Figure 2, blue spots that likely correspond to MB-tagged DiD were also observed inside cells. Our observations indicated also that pDNA were not efficiently delivered into the nucleus. However, it is worth noticing that some spots were close to the nucleus as indicated by their localization in the vicinity of nuclear envelope delimited by Nup153-eGFP.

10 µm

A

The presence of MB inside cells raises the question of their intracellular entry pathway. Do they enter inside cells during the sonoporation process or as a consequence of the sonoporation following their interaction with cells ? The behavior of liposomes based-MB under ultrasonic waves has been studied by predictive models and high speed optical imaging methods [START_REF] Doinikov | Modeling of nonlinear viscous stress in encapsulating shells of lipid-coated contrast agent microbubbles[END_REF][START_REF] Postema | Ultrasonic bubbles in medicine: influence of the shell[END_REF]. Data obtained from studies performed at low acoustic amplitudes have shown that lipid-shelled MB can undergo destruction with microjetting, fragmentation or coalescence under US waves. These responses are dependent on the size, the physico-chemical features of the lipid shell. We performed real time experiments of sonoporation event to investigate how MB behaves towards cells, under our conditions. For that, we developed an experimental set-up combining a color charge coupled device of a PHOTRON FastCam MC-2.1 high-speed camera connected to LSM 500 META Zeiss fluorescence confocal microscope (Figure 4). The transducer was placed in a custom-built sonication chamber, in which an OptiCell ® was mounted. We found that at low mechanical index (< 0.15) at 1 MHz, MB clusters can be formed and pushed within seconds towards cell plasma membrane (not shown). This is in agreement with our recent in vitro data showing that microclusters consisting of lipidencapsulated microbubbles can be formed using primary and secondary radiation forces, and pushed towards vessel walls [START_REF] Kotopoulis | Microfoam formation in a capillary[END_REF]. Interestingly, we also recorded events showing oscillating MB with a small size entered inside cells in few seconds (~10 s) and others that remained stuck on the plasma membrane (Figure 4). Our set up analysis was checked to rule out that the motion of MB takes place in a plane different from that of the cell. This was also validated by fluorescence confocal microscopy analysis. When MB labeled with DiD lipophilic tracer were used, our observations indicated that MB were inside the cell just below the plasma membrane (Figure 4 columns a and b). In summary, data obtained from this study confirmed that pDNA that enters inside cells during sonoporation reaches endocytosis pathway as confirmed by its localization inside late endosomes which are Rab7 positive organelles. A set-up combining high speed camera coupled with confocal fluorescence microscopy has allowed us to observe that some MB were forced to enter the cell during sonoporation whilst other were stuck at the plasma. Altogether our results point out acoustically active microbubbles incorporating the drug inside could release it inside cells.

Figure 1 :

 1 Figure 1: Influence of acoustic pressure on gene transfer efficiency. Cells were sonoporated at US of 1MHz, 40% duty cycle and 60 sec of exposure time in presence of 5 µg of pDNA and 10 7 bubbles. Then, they were further incubated at 37°C during 24h before determining the luciferase activity which is expressed as RLU/mg of proteins. Values shown are means of 2 experiments done in duplicate.

Figure 2 :

 2 Figure 2: Localization of pDNA relative to late endosomes. HeLa Rab7-eGFP cells were sonoporated at optimal acoustic parameters in presence of 2.5 µg of Cy3-tagged pDNA and MB. Green and red staining correspond to Rab7-eGFP positive late endosomes and pDNA-Cy3, respectively. Yellow color corresponds to a co-localization of pDNA and Rab7 (arrows). Confocal microscopy observations were made either immediately after sonoporation (A, B) or 3 hours post-sonoporation (C, D). A and C: phase contrast images; B and D: merge of green and red fluorescence images. White dotted lines in the right frames indicate the cell plasma membrane boundary.

Figure 3 :

 3 Figure 3: Localization of pDNA relative to the nucleus 6h post-sonoporation. Hela Nup153-eGFP cells were sonoporated at optimal acoustic parameters in presence of 2.5 µg of Cy3-tagged pDNA (red) and DiD labelled MB (blue). The green staining corresponds to Nup153-eGFP which delimits the cell nucleus. White dotted lines in the right frames indicate the cell plasma membrane boundary.

Figure 4 :

 4 Figure 4: Experimental setup and a close-up of the fluorescence configuration (top left). A sonoporation event including microbubble internalization during 11 s of sonoporation (right) and selected frames of MB entering a cell (bottom left). Some MB (indicated with the red arrow) entered the cell, whereas others MB stuck to the cell membrane. Lines 1 and 2 represent frames in optical focus from the events of acquisition similar to the right figure before and after sonoporation.


Anthony Delalande

Spiros Kotopoulis
email: s.kotopoulis@2005.hull.ac.uk

Chantal Pichon

Michiel Postema

Cancer cell sonoporation at low acoustic amplitudes

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The use of ultrasound for non-invasive diagnostics in both industry and medical imaging has proven itself to be invaluable due to its low price per examination and ease of use [START_REF] Houze | Measurement of the thickness of thin layers by ultrasonic interferometry[END_REF][START_REF] Tiu | Ultrasound diagnosis of acute appendicitis: imact on cost and outcome in pediatric patients[END_REF][START_REF] Courtney | Factors affecting the ultrasonic intermodulation crack detection technique using bispectral analysis[END_REF][START_REF] Vachhani | Cost effectiveness of ultrasound scan by middle grade doctors in Gynae Assessment Unit (GAU)[END_REF]. ICSV18, 10-14 July 2011, Rio de Janeiro, Brazil 1 18 th International Congress on Sound and Vibration, 10-14 July 2011, Rio de Janeiro, Brazil

In medical-diagnostics, guidelines state an MI<0.3 can be considered safe for pregnant women and neonatals, but yet diagnostic imaging machines allow the use of MI up to 1.9, putting the acoustic intensity used at the examiners discretion. The current regulations are based on the likelihood of inertial cavitation. It is known that inertial cavitation can cause damage not only to cells but also to metals, such as boat propellers and car injectors [START_REF] Palanker | Prevention of tissue damage by water jet during cavitation[END_REF][START_REF] Fuciarelli | Induction of base damage in DNA solutions by ultrasonic cavitation[END_REF][START_REF] Gerr | The Propeller Handbook: The complete reference for choosing, installing, and understanding boat propellers[END_REF][START_REF] Asi | Failure of diesel engine injector nozzel by cavitation[END_REF]. Due to technical challenges, studying the formation and interaction of ultrasound generated cavities is minimal. Therefore the current understanding the consequences of cavitation near or inside cells is limited.

Previous studies on non-invasive, ultrasound-induced therapeutics used acoustic amplitudes corresponding to mechanical indices between 0.2 and 7.0 [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF][START_REF] Pua | Ultrasound-mediated drug delivery[END_REF][START_REF] Bigelow | The destruction of escherichia coli biofilms using high-intensity focussed ultrasound[END_REF].

We analysed the manufacture of efficient, high-frequency, HIFU transducers, capable of highresolution tissue ablation. It was shown that these transducers could be manufactured at low material cost (< £ 25) compared to commercial HIFU PZT transducers, yet these low-budget transducers were capable of generating acoustic amplitudes equivalent to an MI>3.0. Furthermore, single-element high-frequency high-intensity transducers cost even less (< £ 7 in material costs) to manufacture. These transducers were capable of acoustic amplitudes equivalent to an MI=2.7 at a centre transmit frequency of 6.6 MHz, and worked up to the 5 th harmonic of 35 MHz, generating a sound field equivalent to an MI=0.4. These transducers surpassed the safety threshold for diagnostic use even at the 5 th harmonic.

In addition to being economic and time effective, these transducers were also more environmentally friendly when compared to traditional piezo-ceramics, as the piezo-electric crystal used was lead-free and did not require poling.

A limitation in the use of LiNbO 3 as a piezo-electric element is its fragility. The LiNbO 3 elements were seen to be very sensitive to stress concentrations and tended to crack when small physical loads were applied in comparison to PZT piezo-ceramics, e.g., when lapping or dicing to the desired thickness or shape. This fragility was also noticed when applying a high voltage over these elements. For this reason, higher tolerances need to be used when manufacturing transducers with LiNbO 3 active elements. In addition LiNbO 3 is a very poor receiver compared to traditional PZTs due to its low d 33 value, thus it can't be used for imaging and diagnostics.

High-frequency transducers capable of FUS would allow for smaller lesion formation which might surpass the precision of invasive surgery, whilst avoiding the risks associated with invasive surgery [START_REF] Papalois | Surgical Complications: Diagnostics and Treatment[END_REF]. Affordable transducers capable of high-resolution FUS will open a whole new field in ultrasound-induced therapeutics.

We have shown that coagulative necrosis can occur in less than 90 seconds at an MI<2.0 [START_REF] Kotopoulis | Lithium niobate transducer for MRI-guided ultrasonic microsurgery[END_REF], and cellular damage can occur in the presence of microbubbles at an MI<0.2 [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF].

Low-MI ultrasound fields were used to study ultrasound contrast agents in artificial capillaries. We showed that continuous 2.2-MHz and 7.0-MHz ultrasound at an MI<0.015 formed clusters of more than 2000 microbubbles at precise locations. This cluster-formation phenomenon might be used to purposely block vessels, e.g., to temporarily stop blood supply to a tumour, or to gather drug-loaded microbubbles to a specific location for ultrasound-enhanced drug delivery.

The formation of such clusters only occurred at high microbubble concentrations, i.e., at concentrations only theoretically feasible in the human body with undiluted bolus injections. The influence of the flow rate to cluster formation has to be investigated.

To understand the effects of high-intensity ultrasound in tissue, we need to improve our understanding of acoustic cavitation. Acoustic cavitation typically occurs within a few acoustic cycles at unpredictable locations. To study cavitation with high-speed photography, we need to precisely know the site of nucleation. We describe a scientific instrument that is dedicated to this outcome, combining a focussed ultrasound transducer with a pulsed laser [START_REF] Gerold | Laser-nucleated acoustic cavitation in focussed ultrasound[END_REF]. We demonstrated that inertial cavitation can be controllably introduced to the ultrasound focus. Acoustic cavitation was seen to occur at acoustic amplitudes equivalent to an MI=0.7. At higher MI, dynamic cavitation clouds were formed. Our findings will contribute to the understanding of cavitation evolution in focussed ultrasound, including for potential therapeutic applications.

All previous sonoporation publications involved high-MI ultrasound to deliver compounds into cells. Here we explored low-MI methods for drug and gene delivery.

Methodology

An overview of the experimental setup is shown in Figure 1. We have described our experimental setup extensively in Delalande et al. [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF]. In short, a signal consisting of 40 cycles with a centre frequency of 6.6 MHz and a pulse repetition frequency of 10 kHz, was generated by an AFG 3102, dual channel arbitrary function generator (Tektronix, Inc., Beaverton, OR), amplified by a 2100L, 50-dB RF amplifier (Electronics & Innovation Ltd., Rochester, NY) and fed to a custom-built 6.6-MHz ultrasound transducer with a hexagonal Y-36 • lithium niobate element with a maximum diameter of 25 mm [START_REF] Kotopoulis | Lithium niobate transducer for MRI-guided ultrasonic microsurgery[END_REF]. The peak-negative acoustic pressure corresponds to an MI of 0.2. The transducer was placed in a custom-built, 260 × 160 × 150 (mm) 3 Perspex sonication chamber, in which an OptiCell R cell culture chamber (Nunc GmbH & Co. KG, Langenselbold, Germany) was placed. One side of the cell culture chamber contained a monolayer of HeLa cells. Ultrasound contrast agent was injected into the cell culturing chamber before each experiment.

A customised BXFM-F microscope unit with an LCAch N 20×/0.40 NA PhC (Olympus Deutschland GmbH, Hamburg, Germany) and a LUMPlanFL 60×/0.90 NA water-immersion objective (Olympus) was placed on top of the sonication chamber with the objective lens immersed in the water. The colour charge coupled device (CCD) of a PHOTRON FastCam MC-2.1 high-speed camera (VKT Video Kommunikation GmbH, Pfullingen, Germany) was connected to the microscope.

Results and Discussion

Lipid-shelled microbubbles were forced into cells using pulsed ultrasound at MI=0.2 at transmit frequencies of 1.0 MHz and 6.6 MHz. This phenomenon typically takes 2 s from the moment a bubble contacts the cell membrane, to complete dissolution of the gas inside the cell. Most bubble-cell penetration occurred within 8 s from the start of sonication. These results were easily reproducible, independent of the setup geometry. We are the first to observe the translation of entire microbubbles into cells. Since bubbles can be forced into cells, release mechanisms to detach drugs from microbubbles may be of lesser importance. Figure 2 shows an event resampled at 500 Hz. Here a 2 µm bubble penetrates the cancer cell at t =4.404 s, where t =0 s denotes the start of sonication. In Figure 3 shows the same even resampled at 25Hz. The 2 µm bubble completely dissolved 2 s after it penetrated the cell. Targeted drug delivery down to the cellular level, with the use of encapsulated bubbles will allow the use of high-toxicity drugs to be injected into the body, but only delivered to a specific area. Thus, leaving healthy tissue unaffected.

Our sonoporation observations could be attributed to the long pulse lengths used. The bubblecell attraction then may be attributed to secondary Bjerknes forces, similar to those described Kotopoulis and Postema [START_REF] Kotopoulis | Microfoam formation in a capillary[END_REF]. In diagnostic imaging, much shorter pulse lengths are used. Although cells themselves are acoustically active, this acoustic activity is probably negligible to that of microbubbles in high concentrations. Therefore, we expect bubble-cell interaction to be more likely in very low bubble concentrations. This type of bubble-cell attraction is less likely to occur using common clinical diagnostic equipment.

We have shown that it is possible to manufacture low-cost therapeutic transducers. Ultrasound can be used to kill single cancer cells or increase drug uptake. We have managed to induce acoustic cavitation at precise locations

Future work

To ensure reliable performance of LiNbO 3 transducers, several flaws must be addressed. As the Ag-paint electrodes were damaged due to heat and cavitation at the electrode-crystal interface, different electrode materials need to be investigated, e.g., Cr-Au or Ti-Pt. In addition to more reliable electrode application techniques need to be explored. Sputter coating thin film electrodes should eliminate gas pockets at the electrode-crystal interface, leading to better coupling, thus less crystal heating. Other improvements include transducer designs where the natural foci of each active element could be aligned more accurately, lighter support materials, and protective outer layers Our preliminary laser-nucleated acoustic cavitation results show the formation of cavitation clouds at high MI. Very little is known on the dynamics of cavitation clouds. Because clouds are easily induced, their role in FUS must be studied.

We still need to assess the viability of cells penetrated by microbubbles, and subsequently evaluate the suitability of this sonoporation technique for localised drug delivery. This, of course, requires therapeutics to be incorporated in the microbubbles. Although encapsulation processes go beyond the scope of this paper, they are essential to the future success of ultrasound-guided drug and gene delivery. If drug and genes can be successfully coupled to acoustically active vehicles, sonoporation might revolutionise non-invasive therapy as we know it.

Figure 1 .

 1 Figure 1. Experimental setup (top) and a close-up of the sonoporation configuration (bottom).

Figure 2 .Figure 3 .

 23 Figure 2. A sonoporation event resampled at 500 Hz where t =0 s denotes the start of sonication. The 2 µm bubble enters the cell at t =4.404 s. Each frame corresponds to a 20 × 20 (µm) 2 area.

th International Congress on Sound and Vibration, 10-14 July 2011, Rio de Janeiro, Brazil


Spiros Kotopoulis

Anthony Delalande

Chantal Pichon

Michiel Postema

NONLINEAR MICROBUBBLE BEHAVIOUR FOR ENHANCED DRUG UPTAKE

The purpose of this study was to investigate the physical mechanisms of sonoporation, to understand and ameliorate ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. We studied the behaviour of ultrasound contrast agent microbubble near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6.6-MHz ultrasound with a mechanical index of 0.2 and observed using a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell whilst entering. We have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low acoustic amplitude. Hence, if a therapeutic load is added to the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. However, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Introduction

Ultrasound contrast agents are commonly added to the blood stream in ultrasonic imaging: contrast-enhanced ultrasound (CEUS). They consist of microscopically small bubbles (microbubbles) encapsulated by elastic shells. The most common shell materials are phospholipids. During an ultrasound cycle microbubbles oscillate, i.e., they expand and subsequently contract. Depending on their elastic properties, on the local conditions, and on the acoustic settings, they move in the direction of the sound field, coalesce with other microbubbles, fragment, jet, cluster, release their contents, and dissolve in the surrounding liquid [START_REF] Postema | Contrast-enchanced and targeted ultrasound[END_REF]. The diverse behaviour of encapsulated microbubbles in different acoustic regimes has triggered the idea to use them as ultrasound-controlled vehicles to facilitate the delivery of therapeutic agents to a site of interest. Such a noninvasive, localised, sideeffect-free method would revolutionise drug delivery as we know it. Cellular uptake of drugs and deoxyribonucleic acid (DNA) is increased when the region of interest is under sonication, and even more so when an ultrasound contrast agent is present [START_REF] Postema | Ultrasound-directed drug delivery[END_REF]. This increased uptake has been attributed to the formation of transient porosities in the cell membrane that have diameters up to 0.1 µm, i.e., big enough for the transport of drugs into the cell. The pores reseal themselves within one minute. The ultrasound-assisted transient permeabilisation of a cell membrane is called sonoporation. Understanding the physics underlying sonoporation is of uttermost importance for the development of ultrasound-activated therapeutic agents.

Materials and methods

Figure 1 shows the setup used to simultaneously observe and sonicate the cells. A signal consisting of 50 cycles with a centre frequency of 6.6 MHz and a pulse repetition frequency of 10 kHz, i.e. 7.5% duty cycle, was used. The ultrasound was generated by a custom made ultrasound transducer with a hexagonal lithium niobate y-36 • cut active element with a maximum diameter of 25mm. The peak-negative acoustic pressure was measured to be 0.5 MPa in a separate tank and in the sonication chamber itself. This corresponded to an MI of 0.2.

HeLa cancer cells were seeded onto a single side of an OptiCell R cell culture chamber. Ultrasound contrast (MicroMarker TM , VisualSonics B.V., Amsterdam, The Netherlands) agent was injected into the OptiCell R prior to sonication resulting in a dilution of 3.33% v/v. The ultrasound contrast agent was labelled using a DiD [DilC 18 [START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF]] lipophilic probe (Vybrant TM Molecular Probes, Invitrogen, San Diego, CA, USA) to allow microbubble tracking post-sonication. The OptiCell R was then placed into a custom made water bath designed to fit in the x-y stage of a 200M inverted confocal microscope (Carl Zeiss AG, Oberkochen, Germany) coupled to a Axiovert 510 scanning stage (Carl Zeiss AG). Observations were made using a EC Plan-Neofluar 40×/1.30 Oil DIC M27 objective.

A Photron FastCam MC-2.1 high-speed camera (VKT Video Kommunikation GmbH, Pfullingen, Germany) was coupled to the C-mount of confocal fluorescence microscope.

Results

Fluorescence-coated ultrasound contrast agent microbubbles were observed to move into HeLa cells at a low mechanical index (MI) of 0.15, using highspeed video footage under confocal microscopy [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF] an example of which is given in Figure 2. The microbubbles dissolved after entering the cells. The cells survived the sonoporation treatment, confirming the transient character of sonoporation. There are five non-exclusive hypotheses for explaining the sonoporation phenomenon from a physics point of view [START_REF] Postema | CEUS and sonoporation[END_REF]. These have been summarised in Figure 3. It is noted that fragmenting microbubbles cannot create pores in cells, since fragmentation costs energy. (a) Push: During its expansion phase, a microbubble might touch a cell membrane surface. The pushing motion would then cause the cell membrane to be locally disrupted. The pushing mechanism would occur within half an ultrasound cycle. However, under typical sonication conditions, microbubble excursion amplitudes are low. Also, typically, microbubbles are much more flexible than cells. Our current understanding of microbubble physics does not support the push mechanism if the microbubble is not attached. However, if the microbubble is already attached to the cell membrane whilst expanding, pushing under influence of radiation forces might cause the membrane to rupture. The latter mechanism might occur over multiple cycles. (b) Pull: During the contraction phase of an oscillating microbubble, the plasma filling the void left by the contracting bubble might pull the cell membrane towards the microbubble. This pulling motion would then cause the cell membrane to be locally disrupted. The pulling mechanism would occur within half an ultrasound cycle. However, under typical sonication conditions, microbubble contractions do not result in an inertial collapse. Moreover, the replacive mass of a contracting microbubble is much lower than the fluid supposedly causing the pull. Therefore, this mechanism is less plausible than the push mechanism in non-attached conditions. However, if the microbubble is attached to the cell membrane, a similar mechanism might occur as described under (a), where pulling under the influence of radiation forces might cause the membrane to rupture. (c) Jetting: Jetting is the asymmetric collapse of a bubble, creating a funnel-shaped protrusion through the bubble that is directed towards a boundary. This spectacular phenomenon has actually been optically observed on a microscopic scale through cells [START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF]. The jetting phenomenon occurs within half an ultrasound cycle. However, jetting exclusively occurs when using high acoustic amplitudes, at the upper end of clinical settings. Also, there has not been any proof yet of cell survival after jetting. Based on empiric studies that relate jet length to pore size, we excluded the role of jetting as a dominant mechanism involved in sonoporation [6] (d) Streaming: If a microbubble is fixed to a membrane, the fluid streaming around the oscillating bubbles creates enough shear to rupture the membrane [7].

Discussion

Here, long pulse are required to set the fluid in motion. However, low acoustic amplitudes should suffice to create the rupturing shear of a typical cell membrane. Existing research has proven the feasibility of the streaming mechanism in bound conditions, so this mechanism should work for targeted contrast agents that attach to cells. Further research will show whether streaming around weakly-attached microbubbles creates enough shear for cell membranes to rupture. (e) Translation: Owing to radiation forces, lipid-encapsulated microbubbles may translate through cell membranes or channels in the cell membrane. The microbubble may loose part of its shell whilst passing through the cell membrane. As a result, the gas will dissolve after entry. In case of therapeutic loading, the load would be delivered directly into the target cell. This mechanism requires long pulse lengths and low acoustic amplitudes.

Conclusion

All five of these mechanisms require the presence of a microbubble in the vicinity of the cell. Yet, increased drug uptake has also been reported without the use of any ultrasound contrast agent, but merely an ultrasound field itself. Despite speculations about the role of inertial cavitation in the latter, clinical ultrasound scanners do not generate fields strong enough to create such spontaneous microbubbles in blood. Recently, it was found that (bilayer) cell membranes themselves act as cavitation nuclei [8]. Even at modest acoustic pressures, cell membranes might rupture due to the building up of gas cavities inside the bilayer. This important finding would explain the sonoporation in the absence of ultrasound contrast agent microbubbles. Moreover, it explains why bubbles are attracted to cells that are not in the travel path of the ultrasound wave. However, given the low MI at which cells attract microbubbles, more study is required regarding the safe use of ultrasound contrast agents in longpulse-length imaging. In conclusion, in current clinical settings, four non-exclusive mechanisms are plausible for the physical explanation for sonoporation with the aid of ultrasound contrast agent microbubbles.

Figure 1 .

 1 Figure 1. Close up of experimental setup showing configuration for simultaneous sonication and observation using a high-speed camera.

Figure 2 .

 2 Figure 2. Typical event showing two microbubbles translate into a HeLa cell and subsequently dissolving (top) and graphical representation (bottom).

Figure 3 .

 3 Figure 3. Schematic representation of the five mechanisms supposedly involved in sonoporation.

th International Congress on Sound and Vibration, Vilnius, Lithuania, July 8-12,

M. Postema and O. H. Gilja. Jetting does not cause sonoporation. Biomed. Eng., 55:S19-S20, 2010.

th International Congress on Sound and Vibration, Vilnius, Lithuania, July 8-12,

7 P. Marmottant and S. Hilgenfeldt. Controlled vesicle deformation lysis by single oscillating bubbles. Nature, 423:153-156, 2003. 8 B. Krasovitski, V. Frenkel, S. Shoham, and E. Kimmel. Subcellular sonophores: ultrasound induced intramembrane cavitation. Proc. Nat. Acad. Sci., 108(8):3258-3263, 2011.

Acknowledgements

This work has been supported by the Meltzers Høyskolefond.

Bibliography


Anthony Delalande

Spiros Kotopoulis

Michiel Postema

Patrick Midoux

Chantal Pichon
email: chantal.pichon@cnrs-orleans.fr

Sonoporation: mechanistic insights and ongoing challenges for gene transfer

Keywords: ultrasound, microbubbles, physical gene delivery method, gene therapy

come   L'archive ouverte pluridisciplinaire

Introduction

The principle of gene therapy is to introduce gene or nucleic acids into cells to cure genetic deficiencies. The success of gene therapy obtained with the use of viral vectors demonstrates unambiguously the feasibility of this innovative therapy [START_REF] Fischer | Gene therapy of severe combined immunodeficiencies[END_REF][START_REF] Hunter | Toward the construction of integrated physical and genetic maps of the mouse genome using interspersed repetitive sequence PCR (IRS-PCR) genomics[END_REF][START_REF] Nathwani | Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins[END_REF].

To date, viral vectors remain the best vehicles to introduce genes into cells.

Nevertheless, there are still drawbacks inherent to the use of viral molecule observed in gene therapy clinical trials raising serious safety concerns [START_REF] Raper | Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer[END_REF][START_REF] Hacein-Bey-Abina | A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency[END_REF]. In addition, size limitation capacity, cell targeting and manufacturing issues are still difficult to handle despite tremendous progresses made on viral vector bioengineering. Therefore, there is still some room for the development of alternative approaches of high safety, low immunogenicity and easy manufacture. This last decade, many efforts have been done to search for non-viral options. The goal is to design synthetic gene delivery systems that incorporate viral-like features to transfect efficiently cells [START_REF] Mahato | Non-viral peptide-based approaches to gene delivery[END_REF][START_REF] Midoux | Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers[END_REF][START_REF] Wagner | Polylysine-based transfection systems utilizing receptor-mediated delivery[END_REF]. Among non-viral systems, chemical vectors are the most widely used. These vectors have to face on several extracellular and cellular barriers to reach efficiently the target cells.

One of the main challenges is the lack of selectivity towards target tissues explaining their narrow therapeutic index. Lack of specificity causes high toxicity which hampers the efficacy at the target site. For that, designing an efficient targeted delivery strategy is of importance to further improve the delivery systems while reducing side effects. To target chemical vectors, it is possible to couple them with ligands specific to receptors present at the cellular surface of target cells. A second option is to use an externally applied trigger to control the gene delivery in the targeted area. These two strategies are not mutually exclusive and could be combined. Physical trigger can be used either alone or combined with chemical or viral vectors to improve the targeting and/or gene expression efficiency. There are several physical methods starting from hydrodynamic injection to more sophisticated systems such as electroporation-based on electric fields or ultrasound-mediated delivery.

Ultrasound as physical method for delivery

Ultrasound can be used for imaging (ultrasonography) and for physical therapy (pulsed ultrasound mode) [START_REF] Mitragotri | Healing sound: the use of ultrasound in drug delivery and other therapeutic applications[END_REF][START_REF] Lindner | Molecular imaging with contrast ultrasound and targeted microbubbles[END_REF]. These last years, therapeutic applications of ultrasound have gained new interests as a result of its exploitation for drug or gene delivery. Depending on the energy delivered by ultrasound, two types of effects can be produced either thermal or non-thermal each of them having their own application.

High ultrasound intensities produce heating due to the absorption of acoustic energy by tissues; this property is employed in high-intensity focused ultrasound (HIFU) surgery or ultrasound-based physiotherapy. "World Federation for Ultrasound in Medicine and Biology Temperature" has stated that an elevation of 1.5°C is considered safe while an elevation of 4-5°C during 5 minutes could be dangerous [START_REF] Barnett | International recommendations and guidelines for the safe use of diagnostic ultrasound in medicine[END_REF]. At low ultrasound intensities, cavitation and mechanical streaming are the main non-thermal effects obtained. These effects can induce some benefits as tissue healing or ultrasound-mediated delivery. Inertial cavitation is the process of formation, oscillation and collapse of gaseous bubbles driven by an acoustic field [START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF]. The presence of preformed microbubbles in the environment allows reducing the threshold of energy needed for cavitation. Used as external trigger, ultrasound permits to spatiotemporally control the release of drug encapsulated in microbubbles or in their surrounding in a non-invasive manner [START_REF] Kinoshita | Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound[END_REF][START_REF] Frenkel | Ultrasound mediated delivery of drugs and genes to solid tumors[END_REF][START_REF] Kost | Ultrasound-enhanced polymer degradation and release of incorporated substances[END_REF][START_REF] O'neill | Augmentation of targeted delivery with pulsed high intensity focused ultrasound[END_REF][START_REF] Rapoport | Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy[END_REF][START_REF] Schroeder | Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility[END_REF][START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF].

Microbubbles and sonoporation

Microbubbles are gas-filled particles consisting of a gas core encapsulated by a stabilizing shell. They have been first developed as ultrasound contrast agents to differentiate blood and their surroundings under ultrasound due to their low acoustic impedance difference. When microbubbles are driven by ultrasound at a frequency close to their resonance frequency, they oscillate and produce sound [START_REF] Dayton | The magnitude of radiation force on ultrasound contrast agents[END_REF][START_REF] Morgan | Experimental and theoretical evaluation of microbubble behavior: effect of transmitted phase and bubble size[END_REF]. These oscillations lead to an increased permeability of surrounding cells allowing a targeted local drug delivery. The increased cellular uptake has been attributed to the formation of transient pores in the cell membrane facilitating trans-membrane transport of drugs into the cell [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF][START_REF] Zhou | The Size of Sonoporation Pores on the Cell Membrane[END_REF][START_REF] Mehier-Humbert | Plasma membrane poration induced by ultrasound exposure: implication for drug delivery[END_REF][START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF]. This transient permeabilization of a cell membrane is called sonoporation.

Microbubble-cell interactions

Microbubble interactions with cells under ultrasound are the key step for the sonoporation process. To date, five types of interactions have been described (Figure 1). Microbubble oscillations near a plasma membrane could leading to a cell "massage", the "push and pull" phenomena [START_REF] Marmottant | Controlled vesicle deformation and lysis by single oscillating bubbles[END_REF][START_REF] Van Wamel | Micromanipulation of endothelial cells: ultrasoundmicrobubble-cell interaction[END_REF] and microbubble jetting through the plasma membrane [START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF]. This latter is less likely to be the dominant mechanism as shown by in silico studies, in vitro by high-speed optical observations, and in cellulo experiments [START_REF] Postema | Jetting does not cause sonoporation[END_REF]. A microbubble attached to a cell membrane, could also create enough shear to rupture the membrane due to the fluid streaming around the oscillating bubbles. Recently, we have observed a new event: a penetration of microbubble into a cell during sonoporation process under a specific ultrasound setting [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF]. The tracking of fluorescent-labeled microbubbles inside cells after sonoporation has proved this phenomenon. Figure 2 shows two microbubbles (black circle) entering into a cell during ultrasound stimulation, one microbubble was pushed toward the cell deforming the plasma membrane before its cellular penetration. While entering, the microbubble underwent shrinking. This phenomenon took place 6.2 seconds after ultrasound stimulation and 850 ms later, microbubble penetrated inside the cell. This event is slow, compared to the other phenomena described above. Indeed, jetting occurs in the millisecond time scale. Fluorescent microbubbles were observed inside the cells after sonoporation proving their penetration (figure 2C, black arrows). However, it is hard to state if the microbubble has only fused with the plasma membrane or if the structure of the microbubble was preserved after entry.

Nevertheless, these results reinforce the strategy on using microbubbles as drug or gene carriers.

We have also tracked the early interactions between cells and microbubbles at the beginning of the ultrasound stimulation [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF]. In this study, we have used several phospholipid-based microbubbles (SonoVue®, Micromarker TM , Definity®). Before ultrasound stimulation, microbubbles were randomly distributed all around cells.

When ultrasound was turned on, microbubbles immediately interacted with each other forming small clusters (Figure 3) as described by Kotopoulis et al. [START_REF] Kotopoulis | Microfoam formation in a capillary[END_REF]. These clusters could be observed approximately 15 ms after ultrasound application. Interestingly, every cluster present in the field seemed to be attracted to cells and was found at the vicinity of their plasma membrane several seconds after ultrasound stimulation independently of the ultrasound field direction. It is worth noticing that we did not observe such behavior for hard-shelled microbubbles like Quantison™ with the same ultrasound settings. Quantison™ microbubbles interacted also to each other by clustering but they moved only in the direction of the ultrasound field. Since sonoporation is based on microbubble and cell interactions, these observations indicate the importance of microbubble features (composition and acoustic activity). It also arises the question if microbubbles are attracted to specific areas of the plasma which could be worth to be determined.

Cellular effects of ultrasound application

Pore formation has been reported to be essential for sonoporation-mediated delivery [START_REF] Mehier-Humbert | Plasma membrane poration induced by ultrasound exposure: implication for drug delivery[END_REF][START_REF] Mukherjee | Ten-fold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration[END_REF][START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF]. The presence of 100 nm-sized cell membrane pores was observed by electron microscopy and confirmed by flow cytometry with the use of 75 nm-sized fluorescent nanospheres. Using an electro-diffusion model, Zhou and colleagues have evaluated the pore size at 110±40 nm during sonoporation process in the presence of Definity® microbubbles [START_REF] Zhou | The Size of Sonoporation Pores on the Cell Membrane[END_REF]. Concomitant to pore formation, a transient intracellular calcium entrance has been observed during ultrasound application in absence or in presence of microbubbles [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF][START_REF] Juffermans | Ultrasound and microbubble-targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles[END_REF][START_REF] Fan | Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles[END_REF][START_REF] Kumon | Spatiotemporal effects of sonoporation measured by real-time calcium imaging[END_REF][START_REF] Kumon | Ultrasound-induced calcium oscillations and waves in Chinese hamster ovary cells in the presence of microbubbles[END_REF][START_REF] Park | Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells[END_REF][START_REF] Paula | Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis[END_REF][START_REF] Meijering | Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. This intracellular calcium entrance has been supposed to promote the pore closure [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF] and to induce the endocytosis and exocytosis processes [START_REF] Eliasson | Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium[END_REF][START_REF] Macdonald | Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells[END_REF]. The pore closure time has been estimated at 5 seconds after the ultrasound stimulation by flow cytometry and patch clamp experiments [START_REF] Zhou | The Size of Sonoporation Pores on the Cell Membrane[END_REF][START_REF] Mehier-Humbert | Plasma membrane poration induced by ultrasound exposure: implication for drug delivery[END_REF]. Several studies have reported the pore formation after sonoporation, however, it is still not known if these pores are directly responsible of the drug entry. In addition, a production of hydrogen peroxide (H 2 O 2 ) was detected after sonoporation and it seems to be important for the calcium entrance hence for the pore formation [START_REF] Juffermans | Transient permeabilization of cell membranes by ultrasoundexposed microbubbles is related to formation of hydrogen peroxide[END_REF]. Some reports have suggested that this H 2 O 2 production could be related to the cavitation phenomenon [START_REF] Paula | Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis[END_REF][START_REF] Bao | Transfection of a reporter plasmid into cultured cells by sonoporation in vitro[END_REF][START_REF] Miller | Ultrasonic cavitation indirectly induces single strand breaks in DNA of viable cells in vitro by the action of residual hydrogen peroxide[END_REF].

Another cellular effect observed during sonoporation was an hyperpolarization state of cells that can occur in absence [START_REF] Paula | Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis[END_REF] or presence of microbubbles [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF][START_REF] Juffermans | Low-intensity ultrasound-exposed microbubbles provoke local hyperpolarization of the cell membrane via activation of BK(Ca) channels[END_REF][START_REF] Tran | Characterization of cell membrane response to ultrasound activated microbubbles[END_REF]. This hyperpolarization is directly attributed to the opening of the BK Ca channels [START_REF] Juffermans | Low-intensity ultrasound-exposed microbubbles provoke local hyperpolarization of the cell membrane via activation of BK(Ca) channels[END_REF][START_REF] Tran | Characterization of cell membrane response to ultrasound activated microbubbles[END_REF]. It is related to a mechanical stress of the plasma membrane similar to that obtained when applying a mechanical pressure on the membrane using a glass probe [START_REF] Tran | Effect of ultrasound-activated microbubbles on the cell electrophysiological properties[END_REF]. Both hyperpolarization and calcium signaling could increase macromolecules uptake. Besides these phenomena, we have observed an outward transport of small intracellular molecules likely due to membrane destabilization [START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF].

A transient release of small molecules such as enhanced green fluorescent protein (eGFP) was observed from the cytosol of HeLa cells stably expressing eGFP gene while preserving cell viability. These results reinforce the hypothesis of transient pore formation induced by sonoporation.

Cargo size and cellular localization

Sonoporation has been used to deliver drugs in vitro [START_REF] Yoshida | Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells[END_REF][START_REF] Escoffre | Doxorubicin delivery into tumor cells with ultrasound and microbubbles[END_REF] and in vivo [START_REF] Iwanaga | Local delivery system of cytotoxic agents to tumors by focused sonoporation[END_REF].

The presence of microbubbles is a requirement for an efficient delivery. The delivery mechanism by sonoporation appeared to be dependent on the size of the molecules to transfer. The cell membrane permeabilization and viability are highly dependent on the ultrasound parameters used during sonoporation [START_REF] Karshafian | Sonoporation by ultrasound-activated microbubble contrast agents: effect of acoustic exposure parameters on cell membrane permeability and cell viability[END_REF]. Following sonoporation, fluorescent labeled molecules have a different localization profile depending on their molecular size [START_REF] Meijering | Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. Dextran with small size as 3 kDa has a diffuse localization in the cytoplasm and in the nucleus whilst 70 kDa dextran was only found in the cytoplasm.

For dextrans with size over 150 kDa, they were observed as patchy structures localised in the cytoplasm suggesting either aggregates in the cytosol or localisation inside endosomes (Figure 4). After sonoporation, a similar localization was observed for plasmid DNA of 7.5 kb which has an estimated size of approximately 4.95 MDa. These observations suggest a slow plasmid DNA trafficking inside the cells. Taking into account that naked plasmid DNA injected inside the cytosol does not diffuse at all [START_REF] Lukacs | Size-dependent DNA mobility in cytoplasm and nucleus[END_REF]. It is likely that these fluorescent spots corresponded to plasmid DNA located inside endosomes. Overall, these data indicate that pore formation is not the unique mechanism occurring during sonoporation and that endocytosis might also be involved in the uptake [START_REF] Meijering | Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. However, it is still not clear if the type of mechanism(s) involved could be both dependent on the microbubble chemical composition and on the type of tissue insonified.

Optimization of gene delivery

The number of publications relative to the use of sonoporation for gene delivery gives evidence of the potentiality of this method. Ultrasound-enhanced gene delivery has been successfully demonstrated both in vitro and in vivo [START_REF] Newman | Gene therapy progress and prospects: ultrasound for gene transfer[END_REF][START_REF] Pichon | Recent advances in gene delivery with ultrasound and microbubbles[END_REF][START_REF] Suzuki | Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure[END_REF]. The efficacy is dependent on acoustic parameters, the presence of microbubbles and the local concentration of plasmid DNA. Commercial microbubbles as well as customized bubbles lipid-or polymer-shelled have been used in those studies. In most cases, plasmid DNA was not complexed with microbubbles rendering it prone to degradation. As a consequence, the level of gene expression is still not as much as expected even though it is sometimes 1 or 2 orders of magnitude higher than the level obtained with plasmid DNA alone [START_REF] Newman | Gene therapy progress and prospects: ultrasound for gene transfer[END_REF][START_REF] Pichon | Recent advances in gene delivery with ultrasound and microbubbles[END_REF]. Note that it is rather difficult to make comparisons between different reported studies because of the ultrasound set-up and acoustic conditions diversity used by each group.

Ultrasound-enhanced gene transfer has been successful, both with reporter and therapeutic genes in vitro [START_REF] Bao | Transfection of a reporter plasmid into cultured cells by sonoporation in vitro[END_REF][START_REF] Mehier-Humbert | Ultrasound-mediated gene delivery: kinetics of plasmid internalization and gene expression[END_REF][START_REF] Miller | Sonoporation of cultured cells in the rotating tube exposure system[END_REF] and in vivo [START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF][START_REF] Delalande | Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy[END_REF][START_REF] Saito | Sonoporation mediated transduction of pDNA/siRNA into joint synovium in vivo[END_REF][START_REF] Wang | Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents[END_REF]. Biological effects and high spatial targeting of transgene expression were achieved but again not as high as that obtained with electroporation for instance. Still, the specific targeting of the gene expression observed upon local or systemic administration in addition to low toxicity demonstrates the potentiality of this delivery system [START_REF] Lu | Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage[END_REF][START_REF] Shen | Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery[END_REF]. Our recent data showed remarkably that it is possible to get an efficient and sustained gene transfer up to 100 days in Achilles tendons by BR14 lipid shelled MB as shown in figure 6 [START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF][START_REF] Delalande | Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy[END_REF]. Optimized gene transfer was obtained with 1 MHz US frequency, 200 kPa and 40% duty cycle in the presence of 10 µg plasmid DNA and BR 14 microbubbles. The level of gene transfer was 130-fold more than that obtained with naked plasmid DNA.

The level of gene expression obtained here was as good as with adenoviral vectors in tendons highlighting the potential of this system [START_REF] Lou | In vivo gene transfer into tendon by recombinant adenovirus[END_REF][START_REF] Rickert | BMP-14 gene therapy increases tendon tensile strength in a rat model of achilles tendon injury[END_REF].

In addition to acoustic parameters and microbubbles, sonoporation protocols could also be optimized to achieve a good gene transfer. After optimizing acoustic parameters and set-up to transfect HeLa cells, we have assessed if the number of ultrasound applications could improve the gene transfer efficiency since microbubbles interacted with cells and vanished in few seconds. Optimal ultrasound parameters found were 1 MHz frequency, 40% duty cycle, 10 kHz pulse repetition frequency, 150 kPa during 60 seconds in presence of 2.5 µg of plasmid DNA encoding the GFP and 5 µl of Micromarker microbubbles. Gene transfer efficiency was evaluated by flow cytometry 48 hours after sonoporation on HeLa cells. As shown in figure 7, when the ultrasound stimulation was made sequentially in more than one step (the total amount of microbubbles and plasmid DNA being equally dispatched in two or three sequential injections), the gene transfer efficiency was enhanced. Here, the ultrasound stimulation total time was preserved (1×60 sec, 2×30 sec or 3×20 sec). A two-times insonation enhanced by 2.2-fold the gene transfer efficiency (16.2% vs 7.3% for single exposure sonoporation) and a three sequential insonation improved the efficiency to reach 19.9% of GFP expressing cells. This effect could be explained by an improvement of the number of stimulated cells due to a new supply of activable microbubbles. It is worth noticing that the mean fluorescence intensity of transfected cells was not significantly different between all sonoporation conditions validating this hypothesis.

Microbubbles features for efficient gene delivery

While the range of size (mean diameter >1µm) of microbubbles clinically used is appropriate for imaging, they are nevertheless too big and present a polydispersity unsuitable for therapeutic applications [START_REF] Zhao | Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin[END_REF] (for extensive reviews see [START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF][START_REF] Unger | Therapeutic applications of lipid-coated microbubbles[END_REF][START_REF] Ferrara | Driving delivery vehicles with ultrasound[END_REF][START_REF] Huang | Liposomes in ultrasonic drug and gene delivery[END_REF]). Under ultrasound exposure, lipids are compliant to dilatation and compression and this renders gas-filled liposomes more echogenic. The development of acoustically active liposomes has been optimized (for a review [START_REF] Huang | Liposomes in ultrasonic drug and gene delivery[END_REF]) but efforts have been mainly focused to get liposomes bubbles with a high acoustic response. These microbubbles are made of classical phospholipids without any specific feature. Recently, more complexes molecular architecture of liposomes based microbubble have been described: bubbles liposomes that consist of PEGmodified liposomes that encapsulate perfluorocarbon gas enclosed in PEG-lipid micelles [START_REF] Suzuki | Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound[END_REF][START_REF] Suzuki | Effective gene delivery with liposomal bubbles and ultrasound as novel non-viral system[END_REF][START_REF] Un | Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy[END_REF] and a hybrid particle made with microbubble loaded with liposomes that are made of thousands of small unilamellar biotinylated liposomes attached via avidin molecule to biotinylated microbubble [START_REF] Kheirolomoom | Enhanced in vivo bioluminescence imaging using liposomal luciferin delivery system[END_REF].

To achieve improvements in the sonoporation method, the development of new microbubbles able to reach specifically the target and locally deliver the nucleic acid is also needed (figure 8). Targeting microbubbles with antibodies that can recognize a specific antigen present on a cell membrane has allowed microbubble binding even under flow [START_REF] Klibanov | Ultrasound molecular imaging with targeted microbubble contrast agents[END_REF]. For example, microbubbles targeted to the P-selectin and to the single chain VEGF were used to analyze the endothelium inflammation [START_REF] Lindner | Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin[END_REF] and to investigate tumor angiogenesis [START_REF] Anderson | scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis[END_REF], respectively. These microbubbles mainly used in molecular imaging were only based on the acoustic properties and are not able to bind or carry nucleic acids. Some studies have reported the use of cationic microbubbles to directly complex plasmid DNA [START_REF] Anwer | Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration[END_REF][START_REF] Vannan | Ultrasound-mediated transfection of canine myocardium by intravenous administration of cationic microbubble-linked plasmid DNA[END_REF][START_REF] Christiansen | Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles[END_REF][START_REF] Hayashi | Effect of sonoporation on cationic liposome-mediated IFNbeta gene therapy for metastatic hepatic tumors of murine colon cancer[END_REF][START_REF] Tlaxca | Analysis of in vitro transfection by sonoporation using cationic and neutral microbubbles[END_REF]. They consist of gas-made cationic liposomes made with neutral classical phospholipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine ; DMPC or 1,2-distearoyl-sn-glycero-3-phosphocholine ; DSPC) and cationic lipids as (1,2-distearoyl-3-trimethylammoniumpropane). Two main strategies have been proposed to produce microbubbles able to bind nucleic acids: (i) cationic lipids-based microbubble allowing electrostatic interactions between the nucleic acid and microbubbles, (ii) compaction of the nucleic acid using polymers or liposomes linked to the microbubble by biotin-streptavidin interaction [START_REF] Delalande | Ultrasound-assisted gene delivery: recent advances and ongoing challenges[END_REF]. Despite a good acoustic response of these microbubbles, the level of gene transfer obtained in those studies was rather low likely due to different limitations as a large bubble size and the intracellular fate of delivered plasmid DNA. If in addition to pore formation, endocytosis could be also involved in sonoporation, the compartmentalization of therapeutic molecules, and most importantly genes, may affect their efficiency and should be taken into consideration. Therefore, it is necessary to rethink on microbubble composition and to pay more attention on the intracellular fate of both bubbles and their payload.

Conclusion

The non-invasiveness of sonoporation renders it superior to other physical methods as electroporation. The combination of the ultrasound with targeted gas microbubbles as gene carriers holds great promise by offering a double targeting controlling both gene release and gene transfer location [START_REF] Kinoshita | Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound[END_REF][START_REF] Frenkel | Ultrasound mediated delivery of drugs and genes to solid tumors[END_REF][START_REF] Kost | Ultrasound-enhanced polymer degradation and release of incorporated substances[END_REF][START_REF] O'neill | Augmentation of targeted delivery with pulsed high intensity focused ultrasound[END_REF][START_REF] Rapoport | Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy[END_REF][START_REF] Schroeder | Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility[END_REF][START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF]. Nevertheless, there are some challenges that have to be tackled to improve its efficiency. It is important to combine a judicious choice of microbubbles composition and plasmid DNA composition to deliver gene in a specific cell type. So far, cationic microbubbles did not reach the expectations in terms of gene delivery efficiency. This is not surprising because if plasmid DNA is internalized during sonoporation, it has to overcome the same cellular barriers to those encountered by chemical vectors [START_REF] Pichon | Chemical vectors for gene delivery: uptake and intracellular trafficking[END_REF]. Therefore, plasmid DNA must escape from endosomes and be routed inside the nucleus. This could be tackled by fine tuning microbubble composition and reducing their size.

Most importantly, we need to improve our knowledge concerning microbubblecell interactions and how their impact on the gene delivery to fully exploit this method in a safe and efficient way. Since a mechanical stress is induced during ultrasound stimulation, it is of importance to evaluate carefully the impact of this stress on cell metabolism. This would allow us to establish a rational design of sonoporation protocols and to propose sonoporation for possible clinical applications.

Figure 5

 5 Figure 5 shows the localization of fluorescent-labeled plasmid DNA 10, 30 and 45

FiguresFigure 1 .

 1 Figures

  Figure 1. Five mechanisms of a cell membrane pore formation by an oscillating

Figure 2 .

 2 Figure 2. Microbubble entry under sonoporation. (A) Z-stack of fluorescent-labeled

Figure 3 .BFigure 4 .

 34 Figure 3. Microbubble-cell interaction during sonoporation. Observation of

Figure 5 .

 5 Figure 5. Plasmid DNA intracellular trafficking after sonoporation. Fluorescent-

Figure 6 .

 6 Figure 6. Kinetic of luciferase gene expression after sonoporation in mouse Achilles

Figure 7 .Figure 8 . 2 .

 782 Figure 7. Gene transfer efficiency by sonoporation. Sonoporation was performed

Acknowledgements

We are grateful to the Region Centre (Nanodeliv project) the French Agence Nationale de la Recherche (ANR-MEDDU project) for their financial supports.