Anthony Delalande

Michiel Postema

Nathalie Mignet

Patrick Midoux

Chantal Pichon

Ultrasound and microbubble-assisted gene delivery: recent advances and ongoing challenges

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

Gene therapy aims at introducing genetic material into mammalian cells to cure genetic deficiencies and a large variety of acquired diseases. So far, viruses have demonstrated the feasibility of gene therapy and remain the best vehicles to introduce genes into cells. But severe fatal adverse events of viral vectors including acute immune response and insertion mutagenesis observed in gene therapy clinical trials have raised serious safety concerns about their use [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].

Therefore, clinical developments still require the use of alternative approaches of high safety, low immunogenicity and easy manufacture. Moreover, the size limitation capacity, weakness of cell targeting and manufacturing issues has boosted efforts to search for non-viral options. During the last decade, many efforts have been carried out to design synthetic or chemical gene delivery systems that incorporate viral-like features required 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]. The final goal of these systems is to deliver a plasmid encoding a therapeutic protein into target cells upon systemic administration. This challenge could be achieved if several extracellular barriers are overcome to reach the target cells where the therapeutic gene has to be efficiently expressed. Tremendous efforts have been made to improve the efficacy of different types of "synthetic viruses" and elegant strategies have been proposed for that as reported in recent reviews [START_REF] Glover | Artificial Viruses: Exploiting Viral Trafficking for Therapeutics[END_REF][START_REF] Miyata | Rational design of smart supramolecular assemblies for gene delivery: chemical challenges in the creation of artificial viruses[END_REF][START_REF] Wagner | Polymers for siRNA Delivery: Inspired by Viruses to be Targeted, Dynamic, and Precise[END_REF].

However, achieving optimal targeted gene delivery systems based on chemical compounds is still highly challenging. Narrow therapeutic indices and lack of selectivity towards target tissues have been observed. The absence of specificity that causes severe toxicity often hampers the efficacy at the target site. In this respect, innovative delivery strategies that could selectively target tissues of interest are ongoing both in experimental and in clinical research. It is obvious that designing an efficient targeted delivery strategy is the next step to further improve delivery systems and to reduce side effects. Among the delivery systems found are several physical methods starting from a simple naked DNA injection to much more sophisticated systems such as electroporation and ultrasound-mediated delivery (USMD).

Ultrasound is used for diagnostic clinical applications and for treatment either by itself or in combination with drugs [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]. Among the variety of imaging modalities, ultrasonic imaging is a well-established and reliable technique for diagnosis.The development of real-time imaging has allowed for visualizing a variety of anatomical structures noninvasively. These last years, therapeutic applications of ultrasound have gained new interests thanks to the exploitation of ultrasound for delivery. The effect of ultrasound on biological systems was already reported in 1927 [START_REF] Wood | The physical and biological effects of high-frequency soundwaves of great intensity[END_REF], but it took until 1987 until the first study on ultrasound-assisted gene delivery was published [START_REF] Fechheimer | Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading[END_REF]. Depending on the acoustic settings, ultrasound can induce either thermal or non-thermal effects, each having its own applications.

The thermal effects are obtained using high ultrasound intensities and are due to the absorption of acoustic energy by tissue leading to heating. This property of ultrasound is used in techniques like high-intensity focused ultrasound (HIFU) for focussed ultrasound surgery (FUS) or physiotherapy by ultrasound. One of the first applications of HIFU was sonothrombolysis for ischemic stroke treatment. It has been shown that the introduction of microbubbles in the blood pool could act as cavitation nuclei and decrease the energy threshold for thrombolysis by a third [START_REF] Tachibana | Albumin microbubble echo-contrast material as an enhancer for ultrasound accelerated thrombolysis[END_REF]. The thermal effects can present a risk on sonicated tissues; the World Federation for Ultrasound in Medicine and Biology Temperature state that an elevation of 1.5°C is considered safe while an elevation of 4-5°C during 5 minutes is potentially dangerous [START_REF] Barnett | International recommendations and guidelines for the safe use of diagnostic ultrasound in medicine[END_REF]. The non-thermal effects are obtained using lower ultrasound intensities. The main non-thermal effects are inertial cavitation and acoustic streaming. These effects can induce some benefits such as tissue healing or ultrasound-mediated drug or gene delivery.

The use of ultrasound as an external trigger is one approach that has proven to be effective for drug delivery. The drugs to be delivered can be chemotherapeutic agents for cancer therapy applications or oligonucleotides for gene therapy applications. Ultrasound enables to control both the drug release and the release location by use of ultrasound [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][20][START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF].

Ultrasound

Ultrasound is a form of mechanical vibration of matter with a frequency beyond human audible range, i.e., above 20 kHz. The speed of sound is dependent of the medium: roughly 340m/sec in air, 1480 m/sec in pure water, 1550 m/sec in soft tissue and 3700m/sec in bone [START_REF] Lide | Speed of Sound in Various Media[END_REF][START_REF] Goss | Ultrasonic absorption and attenuation in mammalian tissues[END_REF]. The particle excursion in a sound wave is related to the instantaneous local pressure through the wave equation [START_REF] Postema | Fundamentals of Medical Ultrasonics[END_REF]. The wave is damped with propagation distance due to tissue absorption and geometric effects [START_REF] Postema | Fundamentals of Medical Ultrasonics[END_REF]. This behaviour induces mechanical effects in the medium which are amplified when inertial cavitation occurs. Inertial cavitation is the process of formation and subsequent collapse of bubbles driven by an acoustic field [START_REF] Postema | Contrast-enhanced and targeted ultrasound[END_REF]. Acoustic cavitation takes place when the peak-negative pressure amplitude of ultrasound waves at given frequency exceeds the so-called cavitation threshold [START_REF] Postema | Fundamentals of Medical Ultrasonics[END_REF]. If so-called cavitation nuclei brought by impurities in the medium are present, the cavitation threshold is lowered.

Preformed microbubbles serve as cavitation nuclei and are exploited in ultrasonic contrast imaging. These so-called ultrasound contrast agents consist of micron-size gas-filled bubbles (typical diameter ranged is 1-10 µm) encapsulated by an elastic shell. They oscillate during ultrasound exposure (stable cavitation) and demonstrate highly nonlinear behaviour that ameliorates their detection. Oscillating microbubbles introduced in the blood stream produce a sound field themselves and therefore indicate regions of tissue perfusion [START_REF] Morgan | Experimental and theoretical evaluation of microbubble behavior: effect of transmitted phase and bubble size[END_REF][START_REF] Dayton | Optical and acoustical observations of the effects of ultrasound on contrast agents[END_REF]. The acoustic backscattering of these agents is several orders of magnitude higher than the surrounding tissues.

It has been proposed that microbubble activity plays a key role in therapeutic applications [START_REF] Mitragotri | Healing sound: the use of ultrasound in drug delivery and other therapeutic applications[END_REF]. Stable cavitation refers to the (nonviolent) regime of microbubble oscillation, whereas inertial or transient cavitation corresponds to microbubble oscillation with increasingly large amplitudes until collapsing violently [START_REF] Wu | Ultrasound, cavitation bubbles and their interaction with cells[END_REF]. In the nonviolent regime, fluid streaming around oscillating microbubbles may create enough shears to (transiently) open up cell membranes. Furthermore, the microbubbles themselves may interact with, or even translate through cell membranes [START_REF] Postema | Sonoporation: why microbubbles create pores[END_REF]. In the inertial regime, jets from collapsing microbubbles have been observed to penetrate cells, inducing permanent damage [START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF].

Mechanisms of ultrasound-mediated delivery (USMD)

Several studies have been conducted these last years to delineate mechanisms involved in USMD [START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF][START_REF] Duvshani-Eshet | Therapeutic ultrasound optimization for gene delivery: a key factor achieving nuclear DNA localization[END_REF][START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF][START_REF] Mehier-Humbert | Plasma membrane poration induced by ultrasound exposure: implication for drug delivery[END_REF][START_REF] Mehier-Humbert | Ultrasound-mediated gene delivery: kinetics of plasmid internalization and gene expression[END_REF]. Many experimental data have shown that exposure to ultrasound increases the cellular uptake of extracellular molecules. The uptake is significantly higher if an ultrasound contrast agent is added [START_REF] Ferrara | Driving delivery vehicles with ultrasound[END_REF][START_REF] Unger | Therapeutic applications of lipid-coated microbubbles[END_REF]. However, it is under discussion exactly how cells that are subjected to ultrasound internalize extracellular compounds, and which cellular responses they evoke.

Sonoporation is the ultrasound-assisted transient permeabilization of cell membranes. This permeabilization allows for the transfer of molecules between the intra-and extracellular medium. Several sonoporation mechanisms have been identified. The main hypotheses of microbubble interaction with cells are the push and pull mechanisms, microjetting, micro-streaming, and, more recently, translation of microbubbles through cells [START_REF] Postema | Sonoporation: why microbubbles create pores[END_REF]. It has been proposed that the attraction of oscillating microbubbles by cells is caused by sonophores, tiny cavities inside cell membranes [START_REF] Krasovitski | Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects[END_REF].All these sonoporation mechanisms have been demonstrated with the aid of high-speed photography. These mechanisms of membrane poration have been summarized in Figure 1 (inset).

If a microbubble is touching a cell, the microbubble oscillations induce a cellular massage [START_REF] Van Wamel | Micromanipulation of endothelial cells: ultrasound-microbubble-cell interaction[END_REF]. This cellular massage mechanically stimulates the plasma membrane. It has been hypothesised, that during the expansion phase of the microbubble the plasma membrane is pushed inward and that during the compression phase the microbubble pulls the plasma membrane outward [START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF].Repeated push and pull on the plasma membrane creates weaknesses that might lead to the formation of a pore. In a high-mechanical-index regime, the formation of a microjet can occur. A microjet is a fluid protrusion towards a surface during inertial bubble collapse. Jetting has been observed through cells with laser-generated cavities [START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF].

However, this mechanism induces high cell toxicity. Several studies conducted in silico, in vitro, and in cellulo suggest that microbubble jetting behaviour is less likely to be the dominant sonoporation mechanism [START_REF] Postema | Jetting does not cause sonoporation[END_REF].

Another possibility of pore formation mechanism is the rupture of the cell membrane by the streaming flow created by an oscillating microbubble. Indeed, when an oscillating microbubble is attached to a cell membrane the shear forces induced are strong enough to deform and rupture the cell membrane locally [START_REF] Marmottant | Deformation and rupture of lipid vesicles in the strong shear flow generated by ultrasound-driven microbubbles[END_REF][START_REF] Marmottant | Controlled vesicle deformation and lysis by single oscillating bubbles[END_REF].

Recently a hypothesis has been presented for cell membrane pore formation after ultrasonic stimulation in the absence of microbubbles [START_REF] Krasovitski | Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects[END_REF], which involves bilayer sonophores.

In electron microscopy images of fish skin after sonication [START_REF] Frenkel | Ultrasound-induced intercellular space widening in fish epidermis[END_REF], holes in the inter-cellular space were observed, attributed to cavitation between membrane layers (plasma membrane, nucleus and mitochondria). The hypothesis states that cavitation nuclei could be present between the membrane layers inducing a membrane swelling during the rarefaction phase of ultrasound. This deformation could lead to the formation of a pore. This is consistent with the theory on ultrasound-stimulated liposome membranes [START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF].

A new mechanism has been discovered recently by observing microbubble interactions with cells at a low mechanical index (MI). It appears that the microbubbles can enter into the cell straight through the membrane. This translation phenomenon has been proven by the use of fluorescent labelled microbubbles [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF]. The fluorescence of the microbubble has been found in the cell after the observation of a translating microbubble. The microbubble loses (part of) its shell during translation and dissolves quickly after entering the cell. This new mechanism may trigger new drug delivery strategies.

Size of the pore

The formation of a pore during sonication is well-established. Several groups have detected the presence of pores on cell membranes by electron microscopy after sonoporation [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]. Mehier-Humbertet al. were the first to study the poration size. They showed the presence of 100 nm-sized pores in the cell membrane by electron microscopy. The use of fluorescence nanospheres of specific sizes allowing for the measuring of the uptake in MATBIII cells by flow cytometry. They concluded that sonoporation induced a poration of the cell membrane facilitating the transfer of 75-nm-sized spheres. However this study has been done with a specific type of microbubble making the comparison with other studiesdifficult. More recently, Zhou et al. used an electro-diffusion model to measure the size of the pore during sonoporation [START_REF] Zhou | The Size of Sonoporation Pores on the Cell Membrane[END_REF]. This technique based on transmembrane current measurements by voltage clamp [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF] permitted to evaluate the size of the pore in live during sonoporation, the pore size measured was 110±40 nm. This value is consistent with the results obtained by electron microscopy. Although several studies have shown the presence of a pore after sonication, we still do not know if these pores are responsible of the drug entry. The cell membrane permeabilization and viability is highly dependent on the ultrasound parameters [START_REF] Karshafian | Sonoporation by ultrasound-activated microbubble contrast agents: effect of acoustic exposure parameters on cell membrane permeability and cell viability[END_REF]. Juffermans et al. have reviewed the cellular effects of ultrasound [START_REF] Juffermans | Ultrasound and microbubbletargeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles[END_REF]. Ultrasound has been shown to induce a transient intracellular calcium entrance only in the presence of microbubbles [START_REF] Deng | Ultrasound-induced cell membrane porosity[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][53][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 microbubbletargeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. This calcium entrance could promote pore closure [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF] and endocytosis [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 closure time of the pore has been estimated at 5 seconds after ultrasound stimulation [START_REF] Mehier-Humbert | Plasma membrane poration induced by ultrasound exposure: implication for drug delivery[END_REF][START_REF] Zhou | The Size of Sonoporation Pores on the Cell Membrane[END_REF]. It has been reported that sonoporation induces the production of hydrogen peroxide (H 2 O 2 ), which plays an important role in calcium entrance and consequently in pore formation [START_REF] Juffermans | Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide[END_REF]. This production of H 2 O 2 has been shown by others and is thought to be related to inertial cavitation [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]. A hyperpolarisation of sonicated cells has been recorded on different cellular types (Xenopus oocyte, MDA-MB-231, NIH3T3, H9c2) in the 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]and in absence of microbubbles [START_REF] Paula | Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis[END_REF]. This phenomenon is directly related to a mechanical stress of the cell membrane. A similar hyperpolarization was observed applying a mechanical pressure on the membrane using a glass probe [START_REF] Tran | Effect of ultrasoundactivated microbubbles on the cell electrophysiological properties[END_REF]. The hyperpolarization is 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]. The hyperpolarization and the calcium signaling could increase the uptake of macromolecules by endocytosis or macropinocytosis.Moreover, we have found 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. These results reinforce the theory that the pore formation is a transient mechanism. Active sonoporation is not likely to be the only mechanism for increased uptake, because endocytosis mechanism might also be involved [START_REF] Meijering | Ultrasound and microbubbletargeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. 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.

Importance of the size of the cargo and endocytosis

The presence of a pore during sonoporation allows for the passive transfer of molecules between the extracellular and the intracellular medium. Hence, the size of the pore pDNA and clathrin suggesting that pDNA can be routed to the clathrin-mediated endocytosis machinery [START_REF] Meijering | Ultrasound and microbubbletargeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. The link between endocytosis and drug delivery by sonoporation seems to be wellestablished.Yet, when the plasmids reach the endocytosis machinery, they have to escape from the endosomes in order to avoid lysosomal degradation and be available for the import to the nucleus for gene expression. To our knowledge, the trafficking of the plasmid DNA after sonoporation is still ill known.The mechanisms of gene uptake after sonoporation are summarized in Figure 1.

The gene transfer efficiency is dependent on acoustic pressure, frequency, duty cycle, sonication time, the concentration of microbubbles and local plasmid DNA (pDNA), and cell type used [START_REF] Kaddur | Transient transmembrane release of green fluorescent proteins with sonoporation[END_REF][START_REF] Karshafian | Sonoporation by ultrasound-activated microbubble contrast agents: effect of acoustic exposure parameters on cell membrane permeability and cell viability[END_REF][START_REF] Greenleaf | Artificial cavitation nuclei significantly enhance acoustically induced cell transfection[END_REF]. The use of luciferase reporter plasmid has permitted to identify the optimal parameters for an efficient genedeliveryin vitro. Withour experimental setup,the optimal parameters found were 1 MHz, 150kPa, 40% duty cycle (ratio of run time to total cycle time), 60 sec of sonication time and 0.3% (v:v) of microbubble. Under these conditions, less than 10% of cytotoxicity has been measured (Delalande et al., unpublished results).

Microbubbles features for improved efficiency in gene delivery

Most microbubbles used as commercial ultrasound contrast agents have a biodegradable shell composed of phospholipids and a heavy gas core, to prevent them from rapid dissolution. Their diameters are typically below 10 µm, to allow them to pass through the capillary system.

The usual production techniques are based on emulsion by shaking or sonication.

They induce a wide size distribution of the microbubble size. Moreover, the use of ultrasound in this technique generates high pressures and temperatures which could lead to a degradation of the shell components. Getting a narrow distribution of the microbubble population with these methods is challenging and requires the use of centrifugation and filtration steps.

However other microbubble production methods using microfluidic T-junction chambers or coaxial electrohydrodynamic atomisation are under development and could be able to produce microbubbles of a specific diameter size [START_REF] Stride | Novel preparation techniques for controlling microbubble uniformity: a comparison[END_REF].

While the size distributions of microbubbles used is appropriate for imaging, they are nevertheless too wide 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] Ferrara | Driving delivery vehicles with ultrasound[END_REF][START_REF] Unger | Therapeutic applications of lipid-coated microbubbles[END_REF][START_REF] Huang | Liposomes in ultrasonic drug and gene delivery[END_REF]. The thickness and the elasticity of the shell determine microbubbles stability. A microbubble having a very soft shell would be disrupted at small pressure variations. However, a microbubble with a very hard shell would not be able to oscillate. The most elastic shells are made of phospholipids while the stiffer shells are made of polymers or proteins. To reduce the softness of the phospholipid-based microbubble shell, polymers are usually added. Custom microbubbles are mainly made with classical phospholipids, perfluorocarbon gas and pegylated lipids [START_REF] Klibanov | Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging[END_REF][START_REF] Aoi | Herpes simplex virus thymidine kinase-mediated suicide gene therapy using nano/microbubbles and ultrasound[END_REF][START_REF] Lentacker | Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved[END_REF][START_REF] Ibsen | A novel nested liposome drug delivery vehicle capable of ultrasound triggered release of its payload[END_REF]. The presence of the polyethylene glycol (PEG) on the lipid plays the role of emulsifying agent [START_REF] Lee | Coarse-grained model for PEGylated lipids: effect of PEGylation on the size and shape of self-assembled structures[END_REF]. The development of acoustically active microbubbles 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 microbubbles with an optimized acoustic response.

These microbubbles are highly echogenic but again they are made with classical phospholipids without any specific feature. Recently, more complexes molecular architecture of liposome-based microbubble have been described: liposomes bubbles consisting of PEGmodified liposomes that encapsulate perfluoropropane gas enclosed in PEG-lipid micelles [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 | Acoustically-active microbubbles conjugated to liposomes: characterization of a proposed drug delivery vehicle[END_REF]. Of note, the big size, the complexity of architecture and the use of biotin and avidin in those systems are not convenient for in vivo use.

To achieve improvements in sonoporation, the development of new microbubbles able to reach specifically the target and deliver the nucleic acid locally is needed. Targeted microbubbles like Target-Ready Micromarker® (VisualSonics TM ) can be found on the market, the targeting consists of the conjugation of antibodies linked to the microbubble by biotin-streptavidin interaction (Figure 2). Targeted-microbubbles with antibodies can interact with a specific antigen present on a cell membrane allowing the binding of the targeted microbubble even when they are in flow [START_REF] Klibanov | Ultrasound molecular imaging with targeted microbubble contrast agents[END_REF]. For example, microbubbles can be targeted to the P-selectin to analyze the endothelium inflammation [START_REF] Lindner | Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to Pselectin[END_REF] or to the scVEGF have been developed to analyze tumor angiogenesis [START_REF] Anderson | scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis[END_REF]. These microbubbles are mainly used in molecular imaging, their development was only based on the acoustic properties and they are not able to carry nucleic acids.

Two main strategies have been proposed to prepare microbubbles able to bind nucleic acids: (i) the use of cationic lipids (DOPE, DOTAP) in the composition of the microbubble shell allowing electrostatic interactions between the nucleic acid and the microbubble, (ii) Compacting the nucleic acid using polymers or liposomes linked to the microbubble by biotin-streptavidin interaction (Figure 2).The use of cationic microbubbles to directly complex pDNAis being more and more reported [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][83][84]. In these studies, gas-filled cationic liposomes were made with neutral classical (DMPC or DSPC) and cationic lipids as

(1,2-distearoyl-3-trimethylammoniumpropane). There are different manners to couple liposomes or polymers on microbubbles. Amongst them are the deposition of single [START_REF] Lentacker | Ultrasound-responsive polymercoated microbubbles that bind and protect DNA[END_REF]or ii) multi-layers [START_REF] Borden | DNA and polylysine adsorption and multilayer construction onto cationic lipid-coated microbubbles[END_REF]of cationic polymer on the microbubble shell to complex pDNA and a covalent linking of nucleic acids-nanoparticle carriers [START_REF] Vandenbroucke | Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles[END_REF]. These strategies offer the possibility of having the DNA complexes and the microbubble at the same location and likely at the same time. As an example, Sirsiet al. have coupled PEI on the surface of lipid-based microbubbles via polyethylene glycol lipid [START_REF] Sirsi | Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors[END_REF].

Despite a good acoustic response of these microbubbles, the level of in vivo gene transfer obtained with them was rather low may be due to different limitations of the use of large bubbles size (limited or no extravasation). These last years, Maruyama and colleagues have developed bubble liposomes (BLs) which have smaller size (< 1µm) than conventional microbubbles. These structures combine the liposomes features and the acoustic activity of microbubbles. They are composed of DSPC and DSPE-PEG2000-OMe phospholipids filled with perfluoropropane gas [START_REF] Suzuki | Effective in vitro and in vivo gene delivery by the combination of liposomal bubbles (bubble liposomes) and ultrasound exposure. Methods in molecular biology[END_REF]. BLs were efficient to deliver gene in vitro and in vivo. The optimal parameters used for in vitro delivery are 2 MHz ultrasound frequency, duty cycle of 50%, 2.5 W/cm 2 of intensity and 10 sec of exposure time. Whilst for in vivo (solid or ascites tumors), the best parameters are 1 MHz ultrasound frequency, duty cycle of 50%, 1 W/cm 2 of intensity and 1 to 2 min of exposure time [START_REF] Suzuki | Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound[END_REF][START_REF] Suzuki | Effective gene delivery with novel liposomal bubbles and ultrasonic destruction technology[END_REF][START_REF] Suzuki | Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure[END_REF][START_REF] Suzuki | Tumor specific ultrasound enhanced gene transfer in vivo with novel liposomal bubbles[END_REF]. Taking into account the particular features of these BLs (small size, gas particles embedded in liposomes). It will be of interest to know if the different proposed events leading to pore formation can be applied for such structures (figure 1).

Applications of US-mediated delivery for in vivo gene delivery

The potentiality of US-mediated delivery (USMD) applied for gene transfer is attested by the growing number of related publications for both in vitro and in vivo use [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 | Progress in the development of ultrasound-mediated gene delivery systems utilizing nano-and microbubbles[END_REF]. The minimal invasiveness and the high targeting capacity of USMD render the technique potential for clinical transfer. Efficient gene transfer by sonoporation has been obtained when transmitted ultrasound frequencies used are close to those used clinically and extend from 0.5 to 4 MHz. Molecules having variable molecular weights ranging from plasmids [START_REF] Mehier-Humbert | Ultrasound-mediated gene delivery: kinetics of plasmid internalization and gene expression[END_REF],

oligonucleotide [START_REF] Haag | Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours[END_REF] to radioactive tracers [START_REF] Van Wamel | Micromanipulation of endothelial cells: ultrasound-microbubble-cell interaction[END_REF] were successfully incorporated into cells by sonoporation. In the first studies, pDNA were used alone in presence of ultrasound without microbubbles. Significant results have been obtained in vitro as well as in vivo with focused ultrasound [START_REF] Kim | Ultrasound-mediated transfection of mammalian cells[END_REF][99][100]. Microbbubles have been added to enhance the sonoporation efficiency.

However, the level of gene expression is still not higher than that obtained with chemical vectors even though it can be 1 or 2 orders of magnitude more than the level obtained with pDNA 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]. One of the main difficulties in the field of ultrasound-assisted gene delivery is the lack of homogeneity in sonication set-ups and acoustic conditions used yielding comparisons rather hard to make. Indeed, ultrasound parameters applied are variable in terms of frequency (from 1 to 4 MHz), of acoustic power (from to 0.5 to 5 W/cm 2 ) of pulse mode (from 10% to continuous wave) and of stimulation time (from 10 sec to 30 min). The association of ultrasound with gas microbubbles is necessary to get an optimal transfection even though application of ultrasound alone induces a weak molecule transfer into cells [START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF].The majority of studies conducted on USMD has been performed in vitro with a variety of cell types including primary cells [START_REF] Pichon | Recent advances in gene delivery with ultrasound and microbubbles[END_REF]. The main conclusion that can be drawn is the necessity to optimize ultrasound parameters, microbubble and pDNA concentrations for every cell types. Concerning the type of microbubbles, one critical feature is their stability considering the positive correlation between microbubble lifetime and the sonoporation efficiency [101]. Moreover, the stability of gas microbubbles is dependent on both its shell and gas composition [START_REF] Greenleaf | Artificial cavitation nuclei significantly enhance acoustically induced cell transfection[END_REF]101].

These last years, more and more studies report the successful use of ultrasoundenhanced gene transfer in vivo both with reporter and therapeutic genes. In the next step, we will focus more on advances in USMD for in vivo gene delivery applications and especially for cardiovascular applications following systemic injection. Then we will describe studies relative to USMD application following local injection of pDNA in musculoskeletal tissues.

Next, some recent significant data obtained by combining USMD and others delivery systems will be reported.

Cardiovascular applications

It is not surprising that one of the first application of ultrasound and microbubble for gene delivery concerns cardiovascular imaging system. Since, imaging of cardiovascular system has been highly improved by contrast enhanced ultrasound thanks to the wide development of microbubbles in order to detect cardiovascular perfusion pathologies. Taniyama and colleagues have reported a gene therapy trial on rat restenosis after angioplasty model [START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF]. A long-term benefit effect of angioplasty is limited by restenosis phenomenon occurring in 40% of patients. This process is produced by an abnormal smooth muscle cell proliferation of intima. The delivery of pDNA encoding p53 anti-oncogene protein in the presence of ultrasound (2.5 W/cm 2 ) and Optison™ microbubbles has produced an overexpression of p53 in smooth muscle cells leading to an inhibition of intima cells proliferation on rat carotid artery [START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF].

Combination

USMD applications for therapeutic angiogenesis have been widely investigated in myocardial infarction and in hind limb ischemia models in rodents. In a rabbit hind limb ischemic model, the transfer of pDNA encoding the Hepatocyte Growth Factor (HGF) by ultrasound associated with Optison™ microbubbles has allowed to get a better angiogenesis compared to transfer of pDNA alone or pDNA with ultrasound [START_REF] Taniyama | Local delivery of plasmid DNA into rat carotid artery using ultrasound[END_REF]. In a rat model of infarction, a treatment combining ultrasound and microbubbles with pDNA encoding the Ultrasound application away from injection site of pDNA and microbubbles can also induce a sufficient gene delivery. The injection of pDNA encoding the luciferase combined with cationic lipid microbubbles by intra-muscular (IM), intra-venous (IV) or intra-arterial (IA) routes and with an insonation of rat hind limb skeletal muscles has also been assessed [START_REF] Christiansen | Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles[END_REF]. Luciferase activity detected in limb muscles following IA injection was similar to that obtained following IM injection and was 200-fold greater than achieved after IV administration. Overall, these studies demonstrate the strength of this method for future cardiovascular disease therapies.

Bone, muscle, intervertebral discs and tendon

Osteoinduction is required in the field of orthopedics when there are high bone defects caused by fracture, joint related surgery and congenital anomalies. We have also observed such lasting expression in Achilles tendons. Tendons are also composed of tenocytes that divide very slowly. Our recent data show remarkably that it is possible to get an efficient localized gene expression in Achilles tendons which is sustained up to 100 days by using ultrasound and BR14 lipid shelled microbubbles [119,120].

Optimized gene transfer was obtained with 1 MHz ultrasound frequency, 200kPa and 40% duty cycle in the presence of 10µgpDNA and 5×10 5 BR14 microbubbles. The level of gene transfer was 130-fold more efficient than that obtained with naked pDNA. Note that this sustained gene expression is relied on the presence of microbubbles since no effect of ultrasound or microbubble alone was obtained. The level of gene expression obtained here was as good as with adenoviral vectors in tendons highlighting the potential of this system [121]. Most importantly, this approach has permitted the restoration of fibromodulin gene expression fibromodulin KO mice. Ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar that of wild type mice indicating that the fibromodulin expression was enough to restore the collagen fibrils phenotype one week post-transfection.

The effectiveness of this method in tendons could be due to the presence of standing wave which is produced when the reflected ultrasound beam from any sort of interface and the progressive ultrasound beam merge together [150]. Tendons being structures close to bone, a standing wave can be created. Therefore, the exact acoustic power that exactly applied to tendons is hard to know because of the standing wave. This could explain the need of long exposure time as 10 min to achieve high and sustained tendon gene transfer.

Cancer gene therapy

The USMD feasibility has also been examined for cancer gene therapy. Several studies have been performed to evaluate its efficiency on different cancer cells in vitro (for a review see [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]122]). Intra-tumoral injection of anti-tumor gene or systemic injection gene delivery followed by a percutaneous application of ultrasound on the tumor region has also been reported [122-125]. One recent example concerns the production of interleukin (IL)-12

protein through the injection of pDNA encoding IL-12 either in skeletal muscular fibers or directly delivered to the targeted tumor or tissue by blood vessels close to it [START_REF] Suzuki | Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure[END_REF]. Intra-tumor administration is still the best route giving efficient gene expression and in most cases repeated administration is required to reduce the tumor growth [126-128]. One explanation could be the high dilution of pDNA after injection in the blood pool.

From these studies, one can conclude that efficient acoustic conditions have been obtained with 1 MHz with an output of 2 to 5W/cm 2 and 20 to 50% duty cycle with various exposure times that seems to be dependent on the microbubbles used. In most case, in vivo ultrasoundmediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. This efficacy is comparable to that obtained with an electrotransfer-based approach but with the benefit of the non-invasiveness of the method.

Combination of USMD and other delivery systems

USMD has also been used in combination with viral or non-viral vectors in order to enhance gene transfer efficiency. The benefit of this combination is the potentiality to enhance the delivery of the gene in a vicinity of ultrasound and microbubble action. or PEI/pDNA occurred. Moreover, lung hemorrhages were observed with the optimal acoustic parameters. These data suggest the necessity of optimizing the acoustic conditions and the type of microbubbles used to prevent ultrasound bio-effects without altering the benefit on the gene delivery. The main aim will be to develop original microbubbles for safe, non-invasive and target controlled drug delivery by sonoporation in order to enhance the delivery of their payload at a specific location.

Considerations and future perspectives

Exciting results from recent clinical trials demonstrate without doubt the promise of gene therapy. The advances made shows that the field is now surely moving from the conceptual technology to more and more clinical translational. The majority of these advances have been obtained with viral vectors. Therefore, there is still room for non viral methods to be developed since they are more secure in terms of safety. But, improving their efficacy is mandatory for a widespread use. The combination of the ultrasound trigger effect with targeted gas microbubbles as drug/gene carrier 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][20][START_REF] Schroeder | Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes[END_REF]. The noninvasiveness of this system renders it superior to other physical methods as electroporation.

However, some challenges must be overcome to ensure its efficiency and data consistency.

Below are some points that could be considered:

 Rigorous characterization of acoustic fields and description of the experimental configurations: One has noticed that there is lack of consistency regarding the ultrasound exposure configurations in many studies. Researchers in the field are encouraged to describe the set-up used and to make such measurements. This will make the comparison between different studies easier and will help to the identification of critical points which in turn will allow to fast forwarding the technology. Recently, the Safety Committee of the British Medical Ultrasound Society has published a useful set of recommendations that could be easily followed [146].

 Combining a judicious choice of microbubbles composition and plasmid DNA sequence with regard to gene delivery in a specific cell type: It is know well admitted that the gene transfer efficacy of any non viral system is highly dependent on the cell or organ type.

So far in the case of USMD, cationic microbubbles that complex pDNA did not improve the efficiency as much as expected. This could be due to the fact that if an endocytosis process occurs during sonoporation, internalized pDNA has to overcome cellular barriers similar to those encountered by chemical vectors [147]. Therefore, pDNA escape from endosomes and its diffusion and entry inside the nucleus must be improved. This could be tackled by fine tuning microbubble composition and reducing their size.

The composition of pDNA backbone and its length has to be chosen with care depending on the type of gene expression needed (transient or long-term expression). Today, enough experience has been gained in the field of gene delivery to define pDNA constructs that bear specific sequences facilitating nuclear import and long-term gene expression [147][148][149].

 An improved knowledge of microbubble-cell interactions and how they affect the cell and the impact on the gene delivery is still required to exploit this method in a safe and efficient way. A better understanding of underlying mechanisms that are induced at the plasma membrane and inside the cells during the sonoporation would establish a rational determination of ultrasound exposure conditions. Compilation of new observations and identifying molecular signaling mechanisms induced by sonoporation could help to optimize rationally this delivery method.

 The development of ultrasound platform that allow monitoring in real time a concomitant imagine of gene delivery could be useful. In this case, it would be necessary to design original microbubbles that could serve as platforms to develop targeted microbubble for both imaging and delivery applications.

Executive summary:

Gene therapy potentiality to cure inherited and acquired diseases is well established. Nonviral methods offer a good alternative for gene therapy due to safety reasons. Unlike other physical delivery methods, ultrasound-mediated delivery allows to combine the possibility of reaching deeper organs with a non-invasive manner and to restrict the delivery at a specific area.

Sonoporation mechanism

-Sonoporation uses ultrasound activation of microbubbles to increase the permeability of plasma membrane of cells.

-Gas microbubbles oscillations under ultrasound activation induce membrane alteration including formation of transient pores and endocytosis process.

Applications for gene delivery

-Ultrasound and microbubbles-assisted delivery can be exploited to efficiently deliver genes in vitro as well as in vivo in several organs after systemic or local administration. The optimal ultrasound parameters used are highly dependent on the microbubble, the type of cell or the tissue.

-In most case, efficient acoustic conditions have been obtained with 1 MHz with an output ranging from 2 to 5W/cm 2 and from 20 to 50% duty cycle with various exposure times that seems to be dependent on the type of microbubbles used and insonified tissues.

-Ultrasound and microbubble-assisted delivery combined with viral or chemical vectors has a synergetic effect to improve the gene delivery in the vicinity of ultrasound and microbubble action.

Future challenges

-Designing targeted microbubbles that are able to carry the gene of interest could also reduce the amount of nucleic acid needed.

-More knowledge on the impact of microbubbles-cell interactions and the intracellular routing of microbubbles and pDNA has to be gained to fully exploit this methodology. This will allow to fine tune microbubbles composition and to design adequate equipment according to the target organs and to translate the technology for clinical use. functionalization for gene delivery.

  limits the size of the cargo to deliver. The use of fluororescent dextran has shown a completely different localization profile depending of the molecular size of dextran after sonoporation[START_REF] Meijering | Ultrasound and microbubbletargeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. A small dextran molecule (3 kDa) would be localized and diffused inside the cytoplasm and in the nucleus, a medium-sized dextran molecule (70 kDa) would be present in the cytoplasma only, and large dextran molecule (150 and 500 kDa) would be found aspatchy structures in the cytoplasm. Endocytosis plays an important role in the USMD process, indeed it has been shown that endocytosis inhibitors could decrease the drug delivery efficiency by sonoporation[START_REF] Paula | Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis[END_REF][START_REF] Meijering | Ultrasound and microbubbletargeted delivery of macromolecules is regulated by induction of endocytosis and pore formation[END_REF]. In these studies, the authors presented a colocalization of the drug to deliver and the coating endosomes protein, clathrin. Meijering et al. used dextrans as reporter cargos. They observed that the inhibition of endocytosis pathways (clathrin, caveolin and macropinocytosis) inhibited the delivery. The size of pDNA corresponds to the MDa weight range (a 6 kb plasmid average molecular weight would be 3.6 MDa). The important size of plasmid DNA (even folded) implies that the entrance of the plasmid in the cell through a pore is not the main event. Paula et al.have observed a colocalization between

  of the visualization of heart structures and gene delivery has been obtained by exploiting USMD in rats. Commercial and custom microbubbles were tested to deliver pDNA encoding luciferase in the left ventricle [102]. Triggered 1.3 MHz insonation applied every 4 heartbeat gave higher gene expression compared to insonation under continuous mode. The organ specificity is validated by the restriction of gene expression mainly to the heart. The absence of toxicity and the safety of the method (no significant modification in host genes regulation) permit a repeated treatment enhancing the duration of gene expression [103].

  HGF reduced significantly the scar and left ventricle weight with an increased number of capillaries compared to control groups comprising treatment with pDNA alone or pDNA with ultrasound and untreated rats [104]. More recently, the efficiency of USMD was investigated in a severe chronic ischemia model [105]. The treatment consisting of VEGF-165 gene delivery was performed 2 weeks after the induction of unilateral hind limb ischemia in rats. Upon intravenous injection, of 500 µg pDNA encoding VEGF-165 coupled to these microbubbles were injected intravenously (IV), the muscle blood flow assed by contrastenhanced ultrasound and the vessel density investigated by fluorescent microangiography were improved in ischemic adductor muscles exposed to ultrasound and with minimal changes in control groups. The expression of VEGF persisted for 4 weeks. Even though the amount of pDNA used was quite high, this study demonstrates the potentiality of USMD for gene delivery since it was expected that microbubbles would have a low transit through ischemic skeletal muscle. The same research group has compared the efficacy of the VEGF gene delivery by USMD after IV injection to intramuscular (IM) injection [106]. They found that the best increase of the microvascular blood flow and volume was obtained with USMD despite the low transfection efficiency. This superiority could be attributed to the wider localization of transgene expression in the vascular endothelium of capillaries and arterioles of ischemic adductor muscle whilst it was mainly localized to perivascular regions and myocytes of injection site area after IM injection. Along the same line, Fuji and colleagues have delivered VEGF gene or Stem Cell Factor gene, another angiogenic gene, to the murine myocardium seven days after coronary artery ligation [107]. DNA delivery was performed in presence of Definity® (Lantheus medical imaging) MB and US at 8 MHz with a mechanical index of 1.6 directed to the heart during 20 min with an intermittent mode of 1 burst every 500 msec. Two weeks post-treatment, an improved capillary and arterial density, myocardial function and infarct morphometry was obtained in treated animals. The cardiac repair proved by echocardiography and myocardial perfusion, was further improved when multiple treatments were performed (1, 3 or 6 at 2 days interval) [108].

  The improved knowledge of genes involved in bone formation has facilitated the development of new therapeutic applications for bone repair and bone regeneration[109]. Amongst them, Bone Morphogenetic Proteins (BMPs) are known for their ability to induce bone formation. BMP11 transfection has been successfully achieved in canine teeth in vivo by sonoporation, this transfection has allowed a reparative dentin formation[110]. In 2007, a pilot study made by Gazit's group showed that sonoporation is able to induce bone formation in hindlimb muscle of mice[111]. Acoustic parameters consisting of 1 MHz ultrasoundat5 W/cm 2 , 50%duty cycle and 10 min exposure time combined with injection of 50 µg pDNA encoding rhBMP-9 mixed to 5% Optison™ gave the highest efficiency for ectopic bone formation. It is interesting to note that compared to electroporation, the volume of bone formed using sonoporation in muscles was lower but more dense. This difference in the volume density obtained by sonoporation could be attributed to the therapeutic benefit effect of ultrasound. Indeed, it is known that low-intensity pulsed ultrasound (LIPUS) has a positive effect on bone regeneration[112].Another study related to the BMP-2 gene transfer by repeated sonoporation treatments confirmed the promise of USMD for bone induction[113]. BMP-2 has the ability to induce the differentiation of non-osteogenic cells into osteoblasts[114]. Sonoporation has been done at 1 MHz, 4 W/cm 2 and a 50% duty cycle in presence of 75 µg pDNA and SonoVue® microbubbles. X-ray imaging, histochemical analysis and biochemical evaluation were performed to assess the osteo-induction. Data show that when the transcutaneous sonoporationwas repeated 7 times with a 24-hour interval, a cartilage and immature bone were detected in the treated area after 14 days. On day 21, radiographies show denser opacities than on day14 and muscle fibers revealed the presence of bone matrix with bone marrow and many osteoblasts concomitantly with high level of biochemical markers (calcium and alkaline phosphatase). These last decades, the aging population led to an upsurge of age-related pathologies as degenerative diseases. In many spinal disorders, degeneration of the intervertebral discs is an underlying etiology that causes pain and morbidity[115,116].Nishida et al. have been the first who reported the application of USMD on intervertebral discs[117]. They have mixed microbubbles with pDNA encoding the GFP and the firefly luciferase before local injection into rat coccygeal intervertebral discs followed by ultrasound exposure on the surface of injected discs. Ultrasound application induced an improvement of the gene transfer efficiency by 11-fold over pDNA alone. The method has been also successful to transfer siRNA in intervertebral discs[118]. Local injection of siRNA (20pmol) mixed with Optison™ followed by ultrasound exposure (1 MHz, 2 W/cm 2 and 60 sec ultrasound exposure time) has resulted in long-term expression lasting up to 24 weeks. This expression led to long-term downregulation of exogenous reporter gene in rat discs in vivo. This unusual long period of RNA inhibition in intervertebral disc in vivo may be due to the long quiescent state of highly differentiated disc cells.

  An improvement of viral adenoviral gene transfer has been reported in rat cardiomyocytes in vivo following heart exposure with ultrasound. The treatment was consisting of viral particles injection into apical myocardium of the ventricle and application of 1 MHz ultrasound, 1.5 W/cm 2 , continuous wave during 5 min before and after injection. This condition has enhanced the percentage of transfected myocytes from 1.7% to 13.2%[129]. Another study reported by Horward et al .concerns the encapsulation of adenoviral vectors inside Imagent® microbubble injected via tail vein[130]. This has enhanced the specificity of the transgene expression in vitro as well as in vivo to the target organs. Their results elegantly demonstrate that when Imagent® microbubbles were reconstituted in presence of adenoviral vectors, it allows preventing their destruction from complement system activation. The improved transduction in the targeted area was generated upon application of acoustic pressures less than 500kPa.More recently, a combination of adenoviral vector (Ad5 serotype) that encodes for MDR-1 associated with Albumin-coated perfluoropropane gas microbubbles filled with fluorocarbon has been found to be efficient to transduce rabbit bone marrow mononuclear cells[131]. The improvement of gene expression observed was almost 3-fold (8.5% with Ad-5 alone versus 24.5% with Ad-5 combined with ultrasound and microbubble) with no alteration of the cell viability. The presence of microbubbles did not enhance significantly the gene transfer efficiency compared to ultrasound alone. This might be due to the stability of the microbubble used. Indeed, it has been found that microbubble stability is one of the requirements for optimal sonoporation efficiency. A comparison between Optison™, SonoVue® and Sonazoid® has shown that stability, more than size and shell, crucially influenced gene therapy(Alter et al., 2009). Optison™ and Sonazoid® have a similar efficiency in heart that is superior to that of SonoVue®, this latter being less stable. Combining viral vectors with ultrasound and microbubble gives the possibility to downscale by at least one order of magnitude the amount of viral vectors required for an efficient transduction as shown by Muller et al.[132]. In their work, AAV vectors encapsulated in liposomes-based bubbles upon systemic injected have been efficiently targeted into rat hearts upon ultrasound application which is a noninvasive technique in contrast to direct intramyocardial or intracoronary. Of note, all studies have demonstrated that no relevant cardiac adverse events occurred following these treatments [133,134]. Recombinant adeno-associated virus (rAAV) vectors thought being demonstrated as useful tool for gene delivery into eye[135], are hard to scale up rendering their use for clinical applications limited [136]. The easy accessibility of ocular surface has opened the use of physical method as electroporation to transfer therapeutically genes [137]. USMD can deliver pDNA into retinal ganglion cells safely and effectively in vitro and in vivo. An enhancement of rAAV transduction into retinal ganglion cells of rats was obtained after intravitreal injection. Recombinant AAV serotype 2 infection combined with ultrasound has led to an efficient, stable and safe transfection of the retina [138]. Gene transfer in retina using USMD has been tested in vitro and in vivo. The rAAV serotype 2 was chosen to assess the feasibility, efficiency, and safety of the transfection of rAAV2 into RGCs in vivo by USMD. When examining the retinal flat mounts, data prove that eGFP expression in the AAV2-eGFP and USMD-treated group was the strongest and the number of transfected retinal ganglion cells was higher (19.48% versus 3.23%) compared to control groups. Gene transfer into retinal ganglion cells has a tremendous application because of its potentiality to treat glaucomatous optic neuroprotection by preventing apoptosis [139]. A combination of USMD and chemical vectors has been also proposed in different recent studies. Sonoporation has been found to enhance the efficiency of polyethyleneimine (PEI) in vitro [140-142]; as well as in vivo[143,144]. Deshpande and Prausnitz have reported that the combination of ultrasound and PEI has a synergistic effect to increase pDNA transfection efficiency[141]. They examined the influence of ultrasound and PEI:pDNA complexation on transfection of human aortic smooth muscle cells and human prostatic carcinoma DU145 cells with GFP and luciferase reporter genes. Ultrasound stimulation improves the transfection by up to 18-fold relative to naked DNA and by 90-fold when this latter is complexed with PEI. The combination of ultrasound and Optison™ microbubbles with PEI/pDNA complexes increased the transfection up to 200-fold resulting in the transfection of 34% of the cells. Qiu and colleagues have assessed the correlation between acoustic cavitation and sonoporation in ultrasound-mediated gene transfection with PEI in vitro [142]. The study was done on MCF-7 cells and with 1MHz ultrasound frequency in the presence of microbubbles. Data indicate that the transfection efficiency initially increased linearly with the acoustic cavitation, reaching saturation when the acoustic cavitation is too high. There was a high correlation between the measured acoustic cavitation, the sonoporation pore size assessed by electron microscopy, and the cell viability. Xenariou et al. have evaluated the effect of sonoporation on gene delivery mediated by a cationic lipid GL67 complexed with pDNA and PEI/pDNA in lungs [145]. Upon nasal instillation of 100µg of pDNA in mice, their chests were exposed with ultrasound. Despite the possible ultrasound waves attenuation when travelling through air, sonoporation(1 MHz, 3 W/cm 2 , 20% duty cycle and 20 min of exposure time) increases the transfer of naked pDNA when mixed with microbubbles (Optison™), but no enhancement of the transfection efficiency of GL67/pDNA

FiguresFigure 1 .

 1 Figures

Calcium and H 2 O 2

 22 entry enhances the endocytosis process allowing extracellular molecule uptake. Hyperpolarization produced by BK Ca channels activation caused by calcium entry could positively impact the endocytosis. The pDNA was found mainly uptaken via clathrinmediated pathway. The pDNA has to escape from endosomes to avoid lysosomes and it must be imported into the nucleus for its expression.


Véronique Delcroix
email: vdelcroi@uphf.fr

Fatma Essghaier

Kathia Oliveira
email: koliveir@uphf.fr

Philippe Pudlo

Cédric Gaxatte

Francois Puisieux
email: francois.puisieux@chru-lille.fr

Defining an ontology for fall prevention system design

Keywords: knowledge model, fall prevention, ontology

The fall is one of the leading causes of death for elderly. Falls in elderly are caused by multiple factors, such as the aging process itself, persons' behavior, habits and environment. Expert knowledge about all these risk factors is required to provide the right recommendation in order to prevent falls. We have worked on the design of a knowledge-based system. An ontology was defined capturing the core knowledge to be used in the system in order to allow falling risk evaluation and definition of the adapted recommendations.

Introduction

Health systems, in and outside the hospital, must now consider the patient as a central actor of his health. In the context of fall prevention, a large number of stakeholders can be considered, such as physicians, nurses, physiotherapists, close family members, caregivers, and the person herself. Various fall prevention systems have been proposed, often based on sensors [START_REF] Chaccour | From Fall Detection to Fall Prevention : A Generic Classification of Fall-Related Systems[END_REF], but very few are based on a software system. Since expert knowledge is required to provide the right recommendation, a knowledge-based system could help to involve non-expert stakeholders in fall prevention through individual applications that share patient's records together with embedded expert knowledge. This paper proposes an ontology for fall prevention with the core knowledge to support the design of a system that allows an appropriate and constant follow up of the person through a pedagogical and educative approach, including the falling risk evaluation and adapted recommendations. The paper is organized as follows: section 1 present first the context of fall prevention; section 2 presents the design methodology and the proposed ontology; section 3 explores how the ontology contributes to the design of a fall prevention system.

Fall prevention

Today, the population aged 65 and over is very important due to the spectacular increase of life expectancy. Among those persons, one out of three will fall within the year (person living at home), and the problem also occurs in retirement homes and in the hospitals. This frequent event is the first cause of traumatic death in this population and is a multifactorial problem [Haute Autorité de Santé, 2014] et [Bourdessol et Pin, 2008]. The fall risk factors find their roots in the aging process itself, but are also influenced by the person's behavior, habits and environment. Falls can thus be prevented by addressing these risk factors. Fall prevention is achieved through interventions that aim to reduce the risk of falling. Several studies focus on the interest and effects of these interventions [START_REF] Huang | Effects of fall prevention programs for older adults on fall-related injuries: A systematic review and meta-analysis[END_REF]. The possible interventions include: exercise, visual improvement, hazard environmental modification, disease management, education, drug modification, nutritional supplements, health assessment, consultation, referral, assistive devices. Because of the variety of possible interventions, and according to physicians specialized in fall prevention, it is important to select a little number of interventions that are the best adapted to a specific person. Selecting the right recommendations for a given person, according to his/her specific case requires expert knowledge about the fall risk factors and adequate recommendations. Ontologies [Uschold and Grüninger, 1996] are a validated way to provide a general shared knowledge model. We thus propose an ontology to support the design of a software system for fall prevention.

The Ontology for Fall Prevention

The Ontology Design Methodology

To specify the ontology, we followed a methodology (Figure 1) defined based on [START_REF] Uschold | Ontologies: principles, methods and applications[END_REF]Grüninger, 1996] [Noy et al. 2001] composed of four steps: the purpose of the ontology is to support the evaluation of risks of falls in the elderly in order to prevent falls through recommendations. This ontology is the basis for the development of the fall prevention software system. The conceptualization requires the definition of the ontology's scope by setting competence questions (i.e requirements in the form of questions that the ontology must answer) and from that the definition of concepts, relations and constraints (axioms). It represents the knowledge modeling itself. The formalization implied write the ontology with OWL, using Protégé tool. Finally, the validation was done by physicians' analysis and by instantiating the ontology with real cases collected from the historical database of the hospital unit. Aware of that, we organized the ontology in two sub-ontologies: one focused on the elderly person at risk of falling (first two competency questions, presented in section 2.2) and one focused on the recommendations (third competence question). Both sub-ontologies were defined based on the literature, by the interview with specialized physicians on fall prevention, and by the analysis of a historical database from a hospital unit specialized in the treatment of fall for elderly. The conceptualization of the ontology was a long and iterative step of analysis and consensual of terms definition from all these sources with the physicians.

Sub-ontology about Elderly Person at Risk of Falling

Trying to understand the elements that make a person having a risk of fall requires to understand a lot of elements about her pathologies, the medication she takes, her daily activities as well as the environment she lives. Each one of these elements can be refined considering the potential risk it may cause. First of all, the primary attributes of a person (age and sex) are important when evaluating risk factors. Even though these attributes are not considered in an isolated way, they can influence in the evaluation. For instance, women are more likely to fall than men [START_REF] Tinetti | The contribution of predisposing and situational risk factors to serious fall injuries[END_REF]. Moreover, the combined effects of aging and age-related pathologies accentuate the risk of falls [START_REF] Tinetti | Clinical practice: preventing falls in elderly persons[END_REF] and [Bourdessol and Pin, 2008]. In this context, according to [American Geriatrics Society et al., 2001], some chronic or acute disease are known to increase the risk of falling in the elderly.

We organized all the kinds of risk in a taxonomy partially presented in Figure 2. We distinguish between severity factors and incidence factors. Incidence factors are associated with the causes of fall, whereas severity factors are related with the gravity of the consequences of the fall when it occurs, such as injuries and complications. From this taxonomy, we defined associations with the concepts related to the person (see for example the association in Figure 3 about fall risk factors associated with daily life activities).

Exploring Ontology for Fall prevention system Design

The above ontology provides a solid base regarding the identification of important characteristics of the person, fall risk factors and recommendations. In order to provide a system for users who are not expert in fall prevention, some additional aspects have to be considered regarding causal links and correlation between some concepts. For example, alcohol consumption increases the risk of balance impairment. These relationships can be useful when the description of the case to be studied is incomplete. More precisely, the updating of beliefs in unobserved variables given a partial description of the person would allow benefiting from this statistical general knowledge. Ontologies are not designed to model neither causality nor statistical correlations, though several interesting proposals integrate uncertainty in ontology [START_REF] Carvalho | PR-OWL -a language for defining probabilistic ontologies[END_REF]. In order to benefit from that kind of knowledge, we propose using probabilistic graphical models called Bayesian networks [START_REF] Jensen | Bayesian Networks and Decision Graphs[END_REF] since they include probabilistic relationships and allow inference.

The main steps to define a Bayesian network are the variable selection, the graph definition and the parameter learning (local conditional probability distributions). The ontology is of great interest for the first step. The analysis of literature and interviews with experts allows identifying some pieces of information about the structure of the graph. However, more work is required to get a complete definition of a causal graph of a Bayesian network. Finally, data are essential for the learning of the parameters of the probabilistic graphical model, and can also contribute to improve the structure of the graph.

Conclusion

We propose an ontology to support the design of a fall prevention system based on expert knowledge. This knowledge modeling is a fundamental step. Indeed, fall prevention requires a pedagogical approach, meaning that the evaluation of the fall risk and the updating of recommendations to reduce the number of fall risk factors have to be done regularly, involving a large set of non-expert stakeholders. The definition of a probabilistic knowledge model benefits from the ontology, even though it requires different pieces of knowledge. The selection of the variable that must be embedded in the software system is a major step for which the ontology provides answers.

Figure 1 -

 1 Figure 1 -Ontology Design Methodology

Figure 2 -

 2 Figure 2 -Part of the sub-ontology of the elderly at risk of falling