Search

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.

Chemotherapeutic agents such as doxorubicin induce cell cytotoxicity through induction of DNA double-strand breaks. Recent studies have reported the occurrence of DNA double-strand breaks in different cell lines exposed to cavitational ultrasound. As ultrasound stable cavitation can potentiate the therapeutic effects of cytotoxic drugs, we hypothesized that combined treatment with unseeded stable cavitation and doxorubicin would lead to increased DNA damage and would reduce cell viability and proliferation in vitro. In this study, we describe how we determined, using 4T1 murine mammary carcinoma as a model cell line, that unseeded stable cavitation combined with doxorubicin leads to additive DNA double-strand break induction. Combined treatment with doxorubicin and unseeded stable cavitation significantly reduced cell viability and proliferation at 72 h. A mechanistic study of the potential mechanisms of action of the combined treatment identified the presence of cavitation necessary to increase early DNA double-strand break induction, likely mediated by a bystander effect with release of extracellular calcium.

Focused ultrasound are considered to be a promising tool for the treatment of neurological conditions, overcoming the limitations of current neurostimulation techniques in terms of spatial resolution and invasiveness. Much evidence to support the feasibility of ultrasound activation of neurons at the systemic level has already been provided, but to this day, the biophysical mechanisms underlying ultrasound neurostimulation are still widely unknown. In order to be able to establish a clear and robust causality between acoustic parameters of the excitation and neurobiological characteristics of the response, it is necessary to work at the cellular level, or alternatively on very simple animal models. The study reported here responds to three objectives. Firstly, to propose a simple nervous model for the study of the ultrasound neurostimulation phenomenon, associated with a clear and simple experimental protocol. Secondly, to compare the characteristics of this model’s nervous response to ultrasound neurostimulation with its nervous response to mechanical and electrical stimulation. Thirdly, to study the role played by certain acoustic parameters in the success rate of the phenomenon of ultrasound stimulation. The feasibility of generating action potentials (APs) in the giant axons of an earthworm’s ventral nerve cord, using pulsed ultrasound stimuli (f = 1.1 MHz, Ncycles = 175–1150, PRF = 25–125 Hz, Npulses = 20, PA = 2.5–7.3 MPa), was demonstrated. The time of generation (TOG) of APs associated with ultrasound stimulation was found to be significantly shorter and more stable than the TOG associated with mechanical stimulation (p p p

Cavitation is a critical parameter in various therapeutic applications involving ultrasound (US) such as histotripsy, lithotripsy, drug delivery, and cavitation-enhanced hyperthermia. A cavitation exposure outside the region of interest may lead to suboptimal treatment efficacy or in a worse case, to safety issues. Current methods of localizing cavitation are based on imaging approaches, such as beamforming the cavitation signals received passively by a US imager. These methods, although efficient, require expensive equipment, which may discourage potential future developments. We propose a three-hydrophone method to localize the cavitation cloud source. First, the delays between the three receptors are measured by detecting the maximum of their intercorrelations. Then, the position of the source is calculated by either minimizing a cost function or solving hyperbolic equations. After a numerical validation, the method was assessed experimentally. This method was able to track a source displacement with accuracy similar to the size of the cavitation cloud (2-4 mm). This light and versatile method provides interesting perspectives since localization can be executed in real time, and the extension to 3-D localization seems straightforward.

Acoustic cavitation has found a wide range of applications in the last few decades. For potential applications involving cavitation, the acoustic characteristics of a confocal ultrasonic setup are studied: two high-intensity focused ultrasound transducers are mounted so that their focal points overlap. A mathematical simulator is developed that takes into account nonlinear propagation, absorption, and diffraction. Each one of these physical effects is solved in the frequency domain for successive planes. Comparing the confocal setup with equivalent single transducer setups, it is shown that, with the confocal configuration, nonlinear distortion of the waveform is reduced, resulting in a greater peak rarefactional pressure and a lower peak positive pressure. Furthermore, additional features are investigated for confocal configurations such as a greater spatial stability for the focal point, which can be maintained while increasing the pressure level, and a focal region consisting of interference acting as an acoustic trap.

International audience; Doxorubicin, alone or in combination with other anticancer agents, is one of the most widely used chemotherapeutic agents and is administered in a wide range of cancers. However, the use of doxorubicin is limited due to its potential serious adverse reactions. Previous studies have established the ability of high intensity focused ultrasound (HIFU) in combination with various contrast agents to increase intracellular doxorubicin delivery in a targeted and noninvasive manner. In this study, we developed a new sonoporation device generating and monitoring acoustic cavitation bubbles without any addition of contrast agents. The device was used to potentiate the delivery of active doxorubicin into both adherent and suspended cell lines. Combining doxorubicin with ultrasound resulted in a significant enhancement of doxorubicin intracellular delivery and a decrease in cell viability at 48 and 72 h, in comparison to doxorubicin alone. More importantly and unlike previous investigations, our procedure does not require the addition of contrast agents to generate acoustic cavitation and to achieve high levels of doxorubicin delivery. The successful translation of this approach for an in vivo application may allow a significant reduction in the dosage and the adverse effects of doxorubicin therapy in patients.

Summary Targeted and triggered release of liposomal drug using ultrasound (US) induced cavitation represents a promising treatment modality to increase the therapeutic-toxicity ratio of encapsulated chemotherapy. Objectives To study the feasibility and efficacy of a combination of focused US and liposomal doxorubicin (US-L-DOX) release in orthotopic murine models of pancreatic cancer. Material and methods A confocal US setup was developed to generate US inertial cavitation delivery in a controlled and reproducible manner and designed for two distinct murine orthotopic pancreatic cancer models. Controlled cavitation at 1 MHz was applied within the tumors after L-DOX injection according to a preliminary pharmacokinetic study. Results In vitro studies confirmed that L-DOX was cytostatic. In vivo pharmacokinetic study showed L-DOX peak tumor accumulation at 48h. Feasibility of L-DOX injection and US delivery was demonstrated in both murine models. In a nude mouse model, at W9 after implantation (W5 after treatment), US-L-DOX group (median [IQR] 51.43 mm3 [35.1–871.95]) exhibited significantly lower tumor volumes than the sham group (216.28 [96.12–1202.92]), the US group (359.44 [131.48–1649.25]), and the L-DOX group (255.94 [84.09–943.72]), and a trend, although not statistically significant, to a lower volume than Gemcitabine group (90.48 [42.14–367.78]). Conclusion This study demonstrates that inertial cavitation can be generated to increase the therapeutic effect of drug-carrying liposomes accumulated in the tumor. This approach is potentially an important step towards a therapeutic application of cavitation-induced drug delivery in pancreatic cancer.

Due to numerous anatomical and physiological barriers, ocular drug delivery remains a major limitation in the treatment of diseases such as glaucoma, macular degeneration or inflammatory diseases. To date, only invasive approaches provide clinically effective results. Ultrasound can be defined as the propagation of a high-frequency sound wave exposing the propagation media to mechanical and thermal effects. Ultrasound has been proposed as a non-invasive physical agent for increasing therapeutic agent delivery in various fields of medicine. Areas covered: An update on recent advances in transscleral and transcorneal ultrasound-mediated drug delivery is presented. Efficient drug delivery is achieved in vitro, ex vivo and in vivo for various types of materials. Numerous studies indicate that efficacy is related to cavitation. Although slight reversible effects can be observed on the corneal epithelium, efficient drug delivery can be performed without causing damage to the cornea. Expert opinion: Recent developments prove the potential of ultrasound-mediated ocular drug delivery. Cavitation appears to be a preponderant mechanism, opening a way to treatment monitoring by cavitation measurement. Even if no clinical studies have yet been performed, the promising results summarized here are promoting developments toward clinical applications, particularly in assessing the safety of the technique.

Recent preclinical and clinical data suggest that Focused ultrasound (FUS) can enhance host antitumor immune response. FUS is a minimally invasive therapy focusing beams of high power ultrasound to locally induce tissue damage such as thermal necrosis or apoptosis, approved for treatment of some solid malignancies. As a result, there is an interest in combining FUS and immune checkpoint inhibitors (ICI) to enhance ICI response. However, the effect of such combined treatment on the immune response remains unclear. We have evaluated the combination of anti-PD1 and FUS in the MC38 preclinical colon cancer model. FUS was delivered using a confocal device generating and monitoring mechanical cavitational ultrasound (peak negative pressure -20.5MPa). Mice were treated each week with FUS three days before injections of anti-PD1 antibodies. A longitudinal follow-up of tumor growth and overall survival was performed. Lymphoid and myeloid intratumoral immune cells were assessed by flow cytometry on days one, three and six after the start of the combined treatment, including expression levels of co-inhibitory and co-stimulatory molecules such as PD1, PDL-1, TIGIT, LAG3, TIM3 and ICOS. Combined treatment of FUS and ICI led to significant increased survival (up to three-fold for the combination compared to control) and improved tumor growth control. Flow cytometry revealed various changes in lymphoid as well as myeloid populations. The CD8+ TNFa+ / CD4+ FoxP3+ cells ratio was increased for combined group compared to non-treated, the fraction of CD11b+ cells in treated groups was increased and could be explained by an increase in the granulocyte population. Moreover, ICI and FUS combination may lead to increased antigen priming as increased CD11c+ CMHIIhigh conventional dendritic cells (DC), activated CD8- and Sirpa-CD24- subpopulations were found. As for co-inhibitory and co-stimulatory molecules expression at cell surface, flow cytometry showed increased PD1 and LAG3 expression by CD8+ cells on day 1 (respectively two- and three-fold increases for combined group compared to control); an increased TIM3 expression by CD4+ cells for combined group on day 3;an increased PD1 expression by CD4+ cells for FUS-treated groups and by tumor cells for combined group on day 6; increased PDL1 and TIGIT expression by CD8+ cells on day 6. Combination of therapeutic ultrasound and anti PD1 results in a significant improvement in tumor growth control and survival, possibly through significant changes in immune infiltrate such as increased activated CD8+ cells / inhibitory CD4+ cells ratio, an important clinical biomarker of response, increased granulocyte cell populations and an increased DC-mediated response. However, the combined treatment led to increased PD1 expression, as well as of other co-inhibitory molecules, by CD4+, CD8+ and tumor cells possibly explaining the transient effect of the combination. Our observation carries important implications for future FUS/ICI therapies at clinical and mechanistic levels. Citation Format: Cécile Fant, Chloé Grasselly, Morgane Denis, Doriane Mathé, Pierre-Antoine Choffour, Loic Daunizeau, Jean-Louis Mestas, Cyril Lafon, Christophe Caux, Stéphane Depil, Charles Dumontet, Frédéric Padilla. Effect of a combined immune checkpoint inhibitor and mechanical focused ultrasound treatment on intratumoral immune response in a MC38 preclinical model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-078.

Cavitation is a critical parameter in various therapeutic applications involving ultrasound (US) such as histotrispy, lithothripsy, drug delivery, and cavitation-enhanced hyperthermia. A cavitation exposure outside the region of interest may lead to suboptimal treatment efficacy or in a worse case, to safety issues. Current methods of localizing cavitation are based on imaging approaches, such as beamforming the cavitation signals received passively by a US imager. These methods, although efficient, require expensive equipment, which may discourage potential future developments. We propose a threehydrophone method to localize the cavitation cloud source. Firstly, the delays between the three receptors are measured by detecting the maximum of their inter-correlations. Then, the position of the source is calculated by either minimizing a cost function or solving hyperbolic equations. After a numerical validation, the method was assessed experimentally. This method was able to track a source displacement with accuracy similar to the size of the cavitation cloud (2-4 millimeters). This light and versatile method provides interesting perspectives since localization can be executed in real time and the extension to three-dimensional localization seems straightforward.

The combination of liposomal doxorubicin (DXR) and confocal ultrasound (US) was investigated for the enhancement of drug delivery in a rat tumour model. The liposomes, based on the unsaturated phospholipid dierucoylphosphocholine, were designed to be stable during blood circulation in order to maximize accumulation in tumour tissue and to release drug content upon US stimulation. A confocal US setup was developed for delivering inertial cavitation to tumours in a well-controlled and reproducible manner. In vitro studies confirm drug release from liposomes as a function of inertial cavitation dose, while in vivo pharmacokinetic studies show long blood circulation times and peak tumour accumulation at 24-48 h post intravenous administration. Animals injected 6 mg kg(-1) liposomal DXR exposed to US treatment 48 h after administration show significant tumour growth delay compared to control groups. A liposomal DXR dose of 3 mg kg(-1), however, did not induce any significant therapeutic response. This study demonstrates that inertial cavitation can be generated in such a fashion as to disrupt drug carrying liposomes which have accumulated in the tumour, and thereby increase therapeutic effect with a minimum direct effect on the tissue. Such an approach is an important step towards a therapeutic application of cavitation-induced drug delivery and reduced chemotherapy toxicity.

Dioeleoylphosphatidylethanolamine (DOPE)-based liposomes were recently reported as a new class of liposomes for ultrasound (US)-mediated drug delivery. The liposomes showed both high stability and in vitro US-mediated drug release (sonosensitivity). In the current study, in vivo proof-of-principle of US triggered release in tumoured mice was demonstrated using optical imaging. Confocal non-thermal US was used to deliver cavitation to tumours in a well-controlled manner. To detect in vivo release, the near infrared fluorochrome Al (III) Phthalocyanine Chloride Tetrasulphonic acid (AlPcS₄) was encapsulated into both DOPE-based liposomes and control liposomes based on hydrogenated soy phosphatidylcholine (HSPC). Encapsulation causes concentration dependent quenching of fluorescence that is recovered upon AlPcS₄ release from the liposomes. Exposure of tumours to US resulted in a significant increase in fluorescence in mice administered with DOPE-based liposomes, but no change in the mice treated with HSPC-based liposomes. Thus, DOPE-based liposomes showed superior sonosensitivity compared to HSPC-based liposomes in vivo.

Background Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs. Purpose To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model. Methods A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose. Results Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup. Conclusion The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.

Sonodynamic toxicity has always been linked to the cavitation phenomenon. In this work, sonodynamic effect with Photofrin was evaluated with a new ultrasound device: a regulated cavitation generator. In this way, acoustic intensity was substituted with cavitation level as ultrasound parameter. Photofrin potentiated significantly the cavitation cytotoxicity even for low setpoints where no inertial cavitation appeared. Therefore sonodynamic mechanism was principally mechanical, facilitated by the Photofrin insertion in cellular cytoplasmic membranes. This assertion was also supported by the fact that sonodynamic cytotoxicity was independent from the Photofrin presence or absence in the extracellular medium. Reproducible sonodynamic efficiency was perfectly obtained with this new regulated cavitation generator.

Ultrasound cavitation is an essential mechanism involved in the therapeutic local enhancement of drug delivery by ultrasound for cancer treatment. Inertial cavitation also triggers chemical reactions that generate free radicals and subsequent oxidative stress in the tissue. The aim of this study was to measure the oxidative stress induced by inertial cavitation in ex vivo tissue and to test the association between the exposure conditions and the oxidative stress. A confocal ultrasound setup was used to sonicate and create inertial cavitation in freshly excised adipose pig tissue. The ex vivo tissue samples were then processed to measure the quantity of malondialdehyde (MDA), an end-product of polyunsaturated free fatty acid oxidation. The creation of hydroxyterephthalic acid (HTA) from the reaction of terephthalic acid (TA) with free radicals in water was also quantified in vitro . Samples were sonicated for different durations using various amplitudes for the applied pressure. The results showed a minimum 2-fold increase in the amount of detected MDA in the sonicated tissue samples compared to baseline clearly suggesting the generation of free radicals by inertial cavitation. The method exhibited a moderate dependence of MDA generated upon the duration of exposure ( R 2 = 057 , p 0.0001 ) . The average increase in MDA concentration was approximately 2-fold, 5-fold, 6-fold, and 9-fold for exposure durations per unit of volume of 0.13, 0.17, 0.25, and 0.50 s/mm 3 , respectively. The results showed no statistically significant dependence on the amplitude of the pressure within the used range. Both pressure amplitude and exposure duration, however, influenced the HTA concentration ( R 2 > 0.95 , p 0.0001 ) . This biochemical method can be used on ex vivo tissue to detect the generation of free radicals induced by inertial cavitation. In large enough sample populations, the cavitation activity is linked to the exposure conditions of the sonication.

The local application of ultrasound is known to improve drug intake by tumors. Cavitating bubbles are one of the contributing effects. A setup in which two ultrasound transducers are placed confocally is used to generate cavitation in ex vivo tissue. As the transducers emit a series of short excitation bursts, the evolution of the cavitation activity is monitored using an ultrafast ultrasound imaging system. The frame rate of the system is several thousands of images per second, which provides several tens of images between consecutive excitation bursts. Using the correlation between consecutive images for speckle tracking, a decorrelation of the imaging signal appears due to the creation, fast movement, and dissolution of the bubbles in the cavitation cloud. By analyzing this area of decorrelation, the cavitation cloud can be localized and the spatial extent of the cavitation activity characterized.

Ultrasound-induced cavitation has found many applications in the field of cancer therapy. One of its beneficial effects is the enhancement of drug intake by tumor cells. Our group has developed a device that can create and control unseeded cavitation in tissue using ultrasound. We conducted experiments on tumor-bearing mice using our device to assess the impact of sonication on the penetration of fluorescent probes into tumor cells. We studied the influence of pressure level, timing of sonication and sonication duration on treatment efficiency. Our results indicate that fluorescent probes penetrate better into tumors exposed to ultrasound. The best results revealed an increase in penetration of 61% and were obtained when sonicating the tumor in presence of the probes with a peak negative pressure at focus of 19 MPa. At this pressure level, the treatment generated only minor skin damage. Treatments could be significantly accelerated as equivalent enhanced penetration of probes was achieved when multiplying the initial raster scan speed by a factor of four.

Acoustic cavitation can improve local drug delivery in tumors. Without injected external nucleation agents, initiating inertial cavitation requires high negative pressures, which can lead to biological damage. In the present study, unseeded inertial cavitation was obtained in vivo using confocal beams, and the effect of these exposure conditions was assessed on drug structure and activity, shallow tissues and growth of breast tumors. No change was observed in the structure and cytotoxicity of doxorubicin. Experiments were conducted on healthy rats, exposing the thigh and abdomen. Histologic analyses at 72 h and 2 weeks post-treatment demonstrated a modest impact on tissues. Syngeneic 4 T1 breast tumors in mice were sonicated. Immunohistochemical analyses showed that ultrasound did not impact vascular density, proliferation and apoptosis of cancer cells. In addition, ultrasound did not negatively modify cancer cell spreading to the lungs and bone marrow. This provides evidence that these particular parameters can be used safely in vivo.

International audience; Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40–80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

Acoustic cavitation is used for various therapeutic applications such as local enhancement of drug delivery, histotripsy or hyperthermia. One of the utmost important parameter for cavitation creation is the rarefaction pressure. The typical magnitude of the rarefaction pressure required to initiate cavitation from gas dissolved in tissue is beyond the range of the megapascal. Because nonlinear effects need to be taken into account, a numerical simulator based on the Westervelt equation was used to study the pressure waveform and the acoustic field generated by a setup for creation of cavitation consisting of two high intensity focused ultrasound transducers mounted confocally. At constant acoustic power, simulations with only one and both transducers from the confocal setup showed that the distortion of the pressure waveform due to the combined effects of nonlinearity and diffraction is less pronounced when both confocal transducers are used. Consequently, the confocal setup generates a greater peak negat...

Resume L’application d’ultrasons a des liposomes sonosensibles charges de chimiotherapie offre de nouvelles perspectives dans le traitement du cancer. Dans ce travail, des essais in vitro ont permis de definir un indice de cavitation pour permettre le suivi des traitements. Cet indice permet egalement d’evaluer la sonosensibilite de liposomes prototypes. Des experimentations ont ete conduites chez des rats porteurs de tumeurs sous-cutanees. Le temps de 48 heures apres injection a ete choisi pour l’application des ultrasons. Concernant le traitement, les liposomes ralentissent significativement la dynamique tumorale. Les ultrasons combines aux liposomes semblent contribuer au ralentissement du developpement de la tumeur. Cependant, la grande efficacite des liposomes seuls et la possible faible interaction avec les ultrasons ne permettent pas de garantir que l’effet des ultrasons est significatif.

In ocular drug delivery, the sclera is a promising pathway for administering drugs to both the anterior and posterior segments of the eye. Due to the low permeability of the sclera, however, efficient drug delivery is challenging. In this study, pulsed ultrasound (US) was investigated as a potential method for enhancing drug delivery to the eye through the sclera. The permeability of rabbit scleral tissue to a model drug compound, sodium fluorescein, was measured after US-irradiation at 1.1 MHz using time-averaged acoustic powers of 0.5–5.4 W (6.8–12.8 MPa peak negative pressure), with a fixed duty cycle of 2.5% for two different pulse repetition frequencies of 100 and 1000 Hz. Acoustic cavitation activity was measured during exposures using a passive cavitation detector and was used to quantify the level of bubble activity. A correlation between the amount of cavitation activity and the enhancement of scleral permeability was demonstrated with a significant enhancement in permeability of US exposed samples compared to controls. Transmission electron microscopy showed no evidence of significant alteration in viability of tissue exposed to US exposures. A pulsed US protocol designed to maximum cavitation activity may therefore be a viable method for enhancing drug delivery to the eye.

Resume Les generateurs de frequences radiofrequences (RF) sont employes couramment en bloc operatoire. Ils sont generalement utilises comme source d'energie des resecteurs endoscopiques pour l'ablation de l'adenome prostatique. L'etude presentee decrit la physique de l'electrochirurgie avec l'utilisation des courants de frequence elevee pour la coupe et la coagulation, l'interface electrode-tissu d'electrodes dispersives (plaques) et d'electrodes actives (anses) et l'impedance electrique des tissus profonds. Les principales caracteristiques electriques des generateurs RF pour la coupe et l'hemostase sont illustrees par des mesures sur un appareil du commerce. L'energie delivree par le generateur inclut l'energie absorbee par l'interface plaque-peau et l'energie dissipee dans des circuits paralleles associes aux fuites de courant. Les differentes pertes electriques sont susceptibles d'engendrer des risques pour le patient et l'operateur. Les risques rencontres en resection endo-uretrale de la prostate sont recapitules afin d'en assurer un controle optimal.

This work examines the use of lanthanide-based contrast agents and magnetic resonance imaging in monitoring liposomal behavior in vivo. Dysprosium (Dy) and gadolinium (Gd) chelates, Dy-diethylenetriaminepentaacetic acid bismethylamide (Dy-DTPA-BMA) and Gd-DTPA-BMA, were encapsulated in pegylated distearoylphosphatidylethanolamine-based (saturated) liposomes, and then intravenously injected into Copenhagen rats with subcutaneous Dunning AT2 xenografts. Liposome-encapsulated Dy chelate shortens transverse relaxation times (T(2) and T(2)*) of tissue; thus, liposomal accumulation in the tumor can be monitored by observing the decrease in T(2)* relaxation time over time. The tumor was treated at the time of maximum liposomal accumulation (48 h) with confocal, cavitating high-intensity focused ultrasound to induce liposomal payload release. Using liposome-encapsulated Gd chelate at high enough concentrations and saturated liposomal phospholipids induces an exchange-limited longitudinal (T(1)) relaxation when the liposomes are intact; when the liposomes are released, exchange limitation is relieved, thus allowing in vivo observation of payload release as a decrease in tumor T(1).

During recent years, much work has focused on breaking urinary and biliary stones using extracorporeal shock wave devices. Up to now, in terms of fragmentation efficacy, the ‘‘electrohydraulic’’ principle is the gold standard. In an electrohydraulic shock wave generator, a plasma is created by a high‐voltage electrical discharge between two underwater electrodes. This kind of shock wave generator has a significant drawback because of the large variations of pressure pulses. Moreover, the wear of the electrodes is considerable, leading to nonconstant electrical discharge conditions all along the treatment. A new method to canalize the plasma between the electrodes, using highly conductive liquid, was investigated. Based on this principle a new spark plug was designed. This paper deals with the description and the characterization of this new type of spark plug. The electrical and geometrical parameters are presented. Measurements of the focal point pressure amplitude, of the spatial pressure distribution using different voltage settings, are reported. The acoustic energy in the focal zone is computed. We show that using this principle, the wear of the electrodes has been tremendously reduced. A comparison of in vitro results with other shock wave technologies shows that this new spark plug represents a significant breakthrough in terms of stone fragmentation efficacy. This spark plug is not only useful in the medical field, but it should also be of great interest to laboratories.

Applications involving cavitation mechanisms, such as sonoporation, are irreproducible in the case of a fixed‐intensity sonication, due to the non‐stationary behavior of cavitation. We then propose to work at a fixed‐cavitation level instead of under fixed‐intensity sonication conditions. For this purpose a regulated cavitation generator has been developed in a stationary wave field configuration, which allows regulation of the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop based on acoustic cavitation measurements. The cavitation level indicator was quantified by the broadband spectrum noise level relative to inertial cavitation events. This generated inertial cavitation was characterized by both acoustic and chemical measurements, quantifying hydroxyl radicals produced by water sonolysis. While the cavitation level is obtained with a 40% standard deviation for fixed applied acoustic intensities in the range [0.01 3.44] W/cm2, the regulated generator ...

Applications involving acoustic cavitation mechanisms, such as sonoporation, are often poorly reproducible because of the unstationary behavior of cavitation. For this purpose, this study proposes to work at a fixed cavitation level instead of a fixed acoustic intensity. A regulated cavitation generator has been developed in an in vitro configuration of standing wave field. This system implements the regulation of the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop based on acoustic measurements. The experimental setup consists of a plane piezoelectric transducer for sonication (continuous wave, frequency 445 kHz) and a hydrophone pointing to the sonicated medium. The cavitation level is quantified every 5 ms from a spectral analysis of the acoustic signal. The results show that the regulation device generates reproducible mean cavitation levels with a standard deviation lower than 1.6% in the applied intensity range (from 0.12 to 3.44 W/cm 2 ), while this standard deviation can reach 76% without regulation. The feedback loop process imposes precise cavitation level even in low applied acoustic intensity.

The purpose of this study was to define and validate an inertial cavitation dose (CD) based on the detection of broadband noise, designed to monitor ultrasound-mediated drug release from liposomes. The validation consists of using the terephthalate dosimeter to quantify by fluorescence measurements the extent of hydroxyl radical ( OH) production during inertial cavitation. Sonication of samples was performed using tone bursts (pulse repetition frequency (PRF): 10 Hz −1 kHz, duty cycle (dc): 5–25%, Isppa: 4100–12,200 W/cm 2 ) generated by a 1 MHz focused transducer. Three sets of ultrasound parameters with different PRF and dc were selected to be more precisely compared. Results demonstrated an excellent correlation between OH radical production and CD for each set of parameters, but significant differences in hydroxyl radical levels were observed among the sets of parameters. The results were compared with other studies, and the same tendency of variation with pulse duration was demonstrated. Results also showed that the CD was not distorted by peak intensity variations and was a much more reliable indicator than sonication time. Consequently, one validated parameter was selected to monitor drug release from two liposome formulations, and compare their ultrasound sensitivity.

Sonoporation holds many promises in developing an efficient, reproducible and permanent gene delivery vector. In this study, we evaluated sonoporation as a method to transfect nucleic acids in suspension cells, including the human follicular lymphoma cell line RL and fresh human Chronic Lymphocytic Leukemia (CLL) cells. RL and CLL cells were exposed to continuous ultrasound waves (445 kHz) in the presence of either plasmid DNA coding for green fluorescent protein (GFP) or fluorescent siRNA directed against BCL2L1. Transfection efficiency and cell viability were assessed using fluorescent microscopy and flow cytometry analysis, respectively. Knock-down of target protein by siRNA was assessed by immunoblotting. Moreover, sonoporation was used to stably transfect RL cells with a plasmid coding for luciferase (pGL3). These cells were then used for the non-invasive monitoring of tumorigenesis in immunodeficient SCID mice. Sonoporation allows a highly efficient transfection of nucleic acid in suspension cells with a low rate of mortality, both in a tumor cell line and in fresh human leukemic cells. It also allowed efficient transfection of BCL2L1 siRNA with efficient reduction of the target protein level. In conclusion, ultrasound cavitation represents an efficient method for the transfection of cells in suspension, including fresh human leukemic cells.

International audience; Water sonolysis leads to the formation of hydroxyl radicals (OH*). Various techniques are used to detect the OH* production and thus to assess the level of ultrasound-mediated cavitation generated in vitro. In this study, we used terephthalic acid (TA) as an OH* trap. This method is based on the fluorescent properties of hydroxyterephthalic acid (HTA) formed by the reaction of TA with OH* and used as an indicator of the degree of inertial cavitation caused. The experimental system is comprised mainly of a focused piezoelectric ultrasound transmitter and a measurement cell containing 1X PBS/TA diluted solution. In the first part, we aimed to characterize the most appropriate experimental conditions (TA dosimeter solution, irradiation time) in order to optimize the resulting HTA fluorescence values. Then, we could determine that the HTA production increased with the level of the cavitation phenomenon caused by the acoustic power from which OH* production may be estimated.

Sonodynamic cytotoxicity was always linked to the inertial cavitation phenomenon. In this work, sonodynamic effects with Photofrin® were evaluated in controlled cavitation conditions.Photofrin® potentiated significantly the cavitation cytoxicity even for low setpoints where no inertial cavitation appeared. Moreover, the use of antioxidant histidine (10 mM) did not prevent the sonodynamic toxicity when inertial cavitation was preponderant. This was confirmed by mass spectrometry data showing no histidine transformation due to reactive oxygen species during sonodynamic experiments. The results show that sonodynamic mechanism would be principally mechanical, facilitated by the Photofrin® insertion in cellular cytoplasmic membranes.

A technique is described that provides an accurate estimation of the volume of an organ from its ultrasonic cross-sectional images. The technique is applied to two types of ultrasonic investigation, one providing transverse and the other sagittal images. The organ outline has to be traced on each scan. The computer first calculates the area and then the volume from the vector areas and the centroids of a series of sections. The technique has been tested with phantoms of various shapes and volumes made with agar gel. These experiments show that the error in the volume estimation is less than 10% and the variability of measurements is less than 2%.

International audience

In 19 patients, extraction of bile duct stones through the papilla using a Dormia basket or a mechanical lithotripter was not possible following endoscopic sphincterotomy. After the insertion of a nasobiliary drain, extracorporeal lithotripsy was performed with intravenous sedation using an ultrasonographic stone localization system. The number and location of stones were first determined by retrograde cholangiography. At the time of lithotripsy, saline was injected in the bile ducts to modify the acoustic impedance of tissues surrounding the stones, and subsequent ultrasonography was effective in localizing all stones present in 4 of 5 (80%) patients with intrahepatic stones, and 13 of 14 (93%) with common bile duct stones. In 10 patients (53%), fragmentation was satisfactory and the bile ducts were cleared completely. The mean single stone diameter was significantly smaller in successful cases of fragmentation compared with failures (22.8 ± 6.6 mm vs. 40 ± 10 mm). The results in patients with multiple stones were significantly worse than those in patients with single stones of similar size (25% vs. 100% successful fragmentation). Reasons for this difference in results included the small size of the focal area and the reduced ability of ultrasonography (1) to adequately visualize multiple calculi individually and (2) to assess the degree of stone destruction. Care was taken to first await the resolution of infection or the correction of coagulation abnormalities when present; no morbidity following extracorporeal lithotripsy was observed. Despite its 3-step approach (endoscopic sphincterotomy, lithotripsy, and endoscopic extraction), the need for only intravenous sedation and the absence of patient immersion in water render this technique attractive for elderly and frail patients.

Le generateur electrohydraulique utilise en lithotritie est constitue d'un bol ellipsoidal rempli d'eau au foyer duquel sont disposees deux electrodes reliees a un condensateur par l'intermediaire d'un interrupteur. A la fermeture, un arc s'etablit, liberant une energie suffisante pour creer une onde de choc. L'etablissement du regime d'arc est un phenomene peu reproductible induisant des variations importantes de la valeur de la pression generee. Nous montrons que l'utilisation d'un liquide fortement conducteur a la place de l'eau permet de reduire l'ecart type sur les valeurs de pression mesurees de 30 a 2 %. La mesure des parametres electriques suggere que la decharge du condensateur ne se fait plus par l'intermediaire d'un arc mais par l'intermediaire de la resistance formee par le liquide situe entre les electrodes dont la valeur est proche de la resistance critique.

There is growing evidence that acoustic cavitation plays an important role in stone fragmentation during extracorporeal shock wave lithotripsy (ESL) treatment. In addition, side effects of the treatment, such as the hemorrhage and destruction of the tissue in the vicinity of the stone are also ascribed to cavitation phenomenon. Since cavitation is associated with the maximum negative pressure in the shock pulse, it would thus appear that possibility of controlling this pressure would be desirable in ESL applications. This paper describes a novel technique developed to control the ratio of compressional peak (P+) to rarefactional peak pressure (P-) of the shock wave for use in lithotripsy treatment. The procedure is based on the finite amplitude wave generation by focused piezoelectric transducers and subsequent interaction of the shocked waves in the common focal region. The highly asymmetrical shock wave is produced in the focal region by providing an appropriate time delay to each of the high voltage electrical excitation signals which drive the transducers. The degree of relative reduction of negative halfcycles and the corresponding positive halfcycles amplification increases with the number of the acoustic sources used. The practical implementation of the shock wave generator was obtained by using 5 cm diameter, focused 1 MHz transmitter, and additional transducers of identical construction having frequencies corresponding to the harmonics and subharmonics of the 1 MHz frequency. The importance of the results for the future development of lithotripters, and stone treatment efficiency is pointed out.

Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. We developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (peGFP- C1) in adherent and non-adherent cell lines. The frequency spectrum of the signal receive by a hydrophone is used to compute a cavitation index (CI) representative of the inertial cavitation activity. The influence of the CI on transfection efficiency, as well as reproducibility were determined. A real-time feedback loop control on CI was integrated in the process to regulate the cavitation level during sonoporation. In both adherent and non-adherent cell lines, the sonoporation device produced a highly efficient transfection of peGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. Moreover, the sonoporation of non-adherent cell lines Jurkat and K562 was found to be equivalent to nucleofection in terms of efficiency and toxicity while these two cell lines were resistant to transfection with lipofection.

The cavitation noise is a suitable and accurate indicator of the cavitation activity induced in a liquid. Frohly (2000) proposed a quantitative indicator of the transient cavitation calculated as a cavitation spectrum integration in a logarithmic scale and called CNP cavitation noise power indicator. This work studied the relation between a new indicator CMS based on CNP and the exposed-cell mortality in the following conditions. The cell suspension to be insonified (prostatic cells AT2 at 2.5 10/sup 6/ cells/mL) is placed in a medium sample tray (12 wells/tray, 2 mL/well, well diameter: 20 mm). This tray is submerged at mid-depth in degassed water and positioned 5 mm above the face of a flat ultrasonic transducer (diameter 22 mm, frequency: 445.5 kHz; intensity: 0.08-1.09 W/cm/sup 2/, exposure time: 30 sec-4 min). This technical configuration was admitted to be conducive to standing-wave generation through reflection at the air/medium interface in the well thus enhancing the cavitation phenomenon. Laterally to the transducer, a homemade hydrophone (PVDF film of 10 min diameter molded in araldite AY103) was oriented to receive the acoustical signal from the bubbles. From this spectral signal (0.1 to 7.1 MHz bandwidth) recorded every 3 sec on a computer during the exposure condition, CNP was calculated. Its mean value was compared to the cell mortality measured just after ultrasound exposure with a flow cytometer (FACScan) by counting 10000 events. This was accomplished by adding 7AAD solution (Via-Probe kit). Ten exposure conditions were chosen. Two of them corresponded to the experimental limits: no effects on cells (i.e. 0.08 W/cm/sup 2/; 4 min) - complete destruction of cells (i.e. 1.09 W/cm/sup 2/; 30 sec). 3 to 8 measures were realized for each exposure condition. The mortality due to cavitation effect varied from 0.1% to 85.5% and was compared to CMS mean given in relative value (from 3.8% to 97.5%). A simply linear relation between these parameters was given by a correlation coefficient of r/sup 2/=0.81. This correlation coefficient came better when exposed times were considered (for 1 min r/sup 2/=0.82 and for 2 min r/sup 2/=0.95). These results show that CNP is a good indicator of the effect induced by the cavitation phenomenon in cell culture medium in vitro.

A harmonic acoustic wave, fed back by a reflecting target, modifies the electric impedance of the emitter. This effect is studied using a tightly focused beam and various flat targets with known reflection coefficients, which are placed at pressure maxima or minima of the standing wave in the focal zone. It is possible to establish relationships that allow one to determine, for flat targets with unknown acoustic properties, the acoustic impedance and reflection coefficient of the target as well as the acoustic pressure present at the target, only from measurements of the absorbed power and the emitter voltage.

Stable cavitation is produced on the surface of a special target, i.e., an abrasive foil with a grain size of about 15 microm, insonified by a 473 kHz focused beam. Cavitation bubbles are first created by a pressure of about 2 MPa. Progressive reduction of the pressure to about 100 kPa leads to a state of stable cavitation characterized by strong stable emission of the half-order subharmonic. This state can be maintained for five hours if the defining parameters (position and pressure) are optimized and constant to within a few percent. There is strong evidence for the presence of "latent" cavitation bubbles, which can persist for a few minutes without being excited.

In an electrohydraulic generator, two underwater metal electrodes are connected in series with a capacitor charged to a high voltage. When the circuit is switched on, a plasma occurs reaching temperatures of thousands of degrees Celsius and resulting in a compressive pressure pulse. The formation of the plasma is a particular nonreproducible phenomenon inducing great variations in the pressure pulse values. Measurements obtained by triangulation show that the origin of the plasma changes and that the center of the spherical divergent shock wave can be located largely outside the interelectrode gap when tap water is used. However, the center of the shock wave is always located at the same place when electrolyte is used. The plasma duration was also photographed to better study the nonreproducibility of the shock-wave amplitudes. >

Transurethral resection of the prostate is the most common method of relieving urinary outflow obstruction secondary to prostatic enlargement. However, this procedure can be responsible for various complications, including irrigant-fluid absorption and blood loss, both of which are strongly dependent on operation duration time. To reduce the latter, a new resection device has been designed for transurethral prostatectomy. The device basically consists of a rotating cutting loop controlled externally, with three degrees of freedom, to fit the adenoma shape. Its performance is assessed in vitro by drilling conical and semi-ellipsoidal cavities in agar gel models. The mean difference between the calculated and obtained cavity volumes is 3% (SD = 0.9%). The volume cutting rate, found to be independent of the type of cavity drilled, is equal to 2.9 +/- 0.3 cm3 min-1. The advantages of this motorised resection device prototype are reduction in operation duration and accuracy of the resected volume. In vivo resection of a 20 cm3 adenoma in less than 15 min can be expected.