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Microbubble (MB)-assisted ultrasound (US) is an innovative modality for the non-invasive, targeted, and efficient delivery of therapeutic molecules into the brain. Previously, we reported the first metabolomic signature of blood-brain barrier opening (BBBO) induced by MB-assisted US. In the present study, the neurometabolic consequences of acoustically-mediated BBBO on cerebral tissue were investigated using multimodal metabolomics approaches. Sinusoid US waves (1 MHz, peak negative pressure 0.6 MPa, burst length 10 ms, total treatment time 30 s, MB bolus dose 0.7 × 10 5 MBs/g) were applied on the rats' right striatum (ipsilateral side). Brain was collected and both striata were then dissected 3 h, 2 days, and 1 week after BBBO. After tissue preparation, the samples were analyzed using nuclear magnetic resonance spectrometry (NMRS) and high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Our findings showed a slight disruption of metabolic pathways in contralateral striata of animals. Analyses of metabolic pathways indicated changes in amino acid metabolisms. In addition, tryptophan derivate dosages revealed the perturbation of a central metabolite of the kynurenine pathway (i.e., 3-hydroxy-kynurenine). In conclusion, the acoustically-mediated BBBO of the ipsilateral cerebral hemisphere induced significant change in metabolism of contralateral one., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Presset, Bodard, Lefèvre, Millet, Oujagir, Dupuy, Iazourène, Bouakaz, Emond, Escoffre and Nadal-Desbarats.)

Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.

The blood-brain barrier (BBB) maintains brain homeostasis, regulates influx and efflux transport, and provides protection to the brain tissue. Ultrasound (US) and microbubble (MB)-mediated blood-brain barrier opening is an effective and safe technique for drug delivery in-vitro and in-vivo. However, the exact mechanism underlying this technique is still not fully elucidated. The aim of the study is to explore the contribution of transcytosis in the BBB transient opening using an in-vitro model of BBB. Utilizing a diverse set of techniques, including Ca 2+ imaging, electron microscopy, and electrophysiological recordings, our results showed that the combined use of US and MBs triggers membrane deformation within the endothelial cell membrane, a phenomenon primarily observed in the US + MBs group. This deformation facilitates the vesicles transportation of 500 kDa fluorescent Dextran via dynamin-/caveolae-/clathrin- mediated transcytosis pathway. Simultaneously, we observed increase of cytosolic Ca 2+ concentration, which is related with increased permeability of the 500 kDa fluorescent Dextran in-vitro. This was found to be associated with the Ca 2+ -protein kinase C (PKC) signaling pathway. The insights provided by the acoustically-mediated interaction between the microbubbles and the cells delineate potential mechanisms for macromolecular substance permeability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Treating pathologies of the inner ear is a major challenge. To date, a wide range of procedures exists for administering therapeutic agents to the inner ear, with varying degrees of success. The key is to deliver therapeutics in a way that is minimally invasive, effective, long-lasting, and without adverse effects on vestibular and cochlear function. Microbubble-assisted ultrasound ("sonoporation") is a promising new modality that can be adapted to the inner ear. Combining ultrasound technology with microbubbles in the middle ear can increase the permeability of the round window, enabling therapeutic agents to be delivered safely and effectively to the inner ear in a targeted manner. As such, sonoporation is a promising new approach to treat hearing loss and vertigo. This review summarizes all studies on the delivery of therapeutic molecules to the inner ear using sonoporation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tumor spheroids as well as multicellular tumor spheroids (MCTSs) are promising 3D in vitro tumor models for drug screening, drug design, drug targeting, drug toxicity, and validation of drug delivery methods. These models partly reflect the tridimensional architecture of tumors, their heterogeneity and their microenvironment, which can alter the intratumoral biodistribution, pharmacokinetics, and pharmacodynamics of drugs. The present review first focuses on current spheroid formation methods and then on in vitro investigations exploiting spheroids and MCTS for designing and validating acoustically mediated drug therapies. We discuss the limitations of the current studies and future perspectives. Various spheroid formation methods enable the easy and reproducible generation of spheroids and MCTSs. The development and assessment of acoustically mediated drug therapies have been mainly demonstrated in spheroids made up of tumor cells only. Despite the promising results obtained with these spheroids, the successful evaluation of these therapies will need to be addressed in more relevant 3D vascular MCTS models using MCTS-on-chip platforms. These MTCSs will be generated from patient-derived cancer cells and nontumor cells, such as fibroblasts, adipocytes, and immune cells.

Sonoporation using microbubble-assisted ultrasound increases the permeability of a biological barrier to therapeutic molecules. Application of this method to the round window membrane could improve the delivery of therapeutics to the inner ear. The aim of this study was to assess the safety of sonoporation of the round window membrane in a sheep model. To achieve this objective, we assessed auditory function and cochlear heating, and analysed the metabolomics profiles of perilymph collected after sonoporation, comparing them with those of the control ear in the same animal. Six normal-hearing ewes were studied, with one sonoporation ear and one control ear for each. A mastoidectomy was performed on both ears. On the sonoporation side, Vevo MicroMarker ® microbubbles (MBs; VisualSonics-Fujifilm, Amsterdam, The Netherlands) at a concentration of 2 × 10 8 MB/mL were locally injected into the middle ear and exposed to 1.1 MHz sinusoidal ultrasonic waves at 0.3 MPa negative peak pressure with 40% duty cycle and 100 μs interpulse period for 1 min; this was repeated three times with 1 min between applications. The sonoporation protocol did not induce any hearing impairment or toxic overheating compared with the control condition. The metabolomic analysis did not reveal any significant metabolic difference between perilymph samples from the sonoporation and control ears. The results suggest that sonoporation of the round window membrane does not cause damage to the inner ear in a sheep model.

Recent technological developments in ultrasound (US) imaging and ultrasound contrast agents (UCAs) have improved diagnostic confidence in echography. In the clinical management of melanoma, contrast-enhanced ultrasound (CEUS) imaging complements conventional US imaging (i.e., high-resolution US and Doppler imaging) for clinical examination and therapeutic follow-up. These developments have set into motion the combined use of ultrasound and UCAs as a new modality for drug delivery. This modality, called sonoporation, has emerged as a non-invasive, targeted and safe method for the delivery of therapeutic drugs into melanoma. This review focuses on the results and prospects of using US and UCAs as dual modalities for CEUS imaging and melanoma treatment., Competing Interests: Conflict of interest disclosure The authors have no conflicts of interest to disclose., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that still lacks an efficient therapy. The barriers between the central nervous system (CNS) and the blood represent a major limiting factor to the development of drugs for CNS diseases, including ALS. Alterations of the blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) have been reported in this disease but still require further investigations. Interestingly, these alterations might be involved in the complex etiology and pathogenesis of ALS. Moreover, they can have potential consequences on the diffusion of candidate drugs across the brain. The development of techniques to bypass these barriers is continuously evolving and might open the door for personalized medical approaches. Therefore, identifying robust and non-invasive markers of BBB and BSCB alterations can help distinguish different subgroups of patients, such as those in whom barrier disruption can negatively affect the delivery of drugs to their CNS targets. The restoration of CNS barriers using innovative therapies could consequently present the advantage of both alleviating the disease progression and optimizing the safety and efficiency of ALS-specific therapies.

Microbubble (MB)-assisted ultrasound (US) is a promising physical method to increase non-invasively, transiently, and precisely the permeability of the blood-brain barrier (BBB) to therapeutic molecules. Previous preclinical studies established the innocuity of this procedure using complementary analytical strategies including transcriptomics, histology, brain imaging, and behavioral tests. This cross-sectional study using rats aimed to investigate the metabolic processes following acoustically-mediated BBB opening in vivo using multimodal and multimatrices metabolomics approaches. After intravenous injection of MBs, the right striata were exposed to 1-MHz sinusoidal US waves at 0.6 MPa peak negative pressure with a burst length of 10 ms, for 30 s. Then, the striata, cerebrospinal fluid (CSF), blood serum, and urine were collected during sacrifice in three experimental groups at 3 h, 2 days, and 1 week after BBB opening (BBBO) and were compared to a control group where no US was applied. A well-established analytical workflow using nuclear magnetic resonance spectrometry and non-targeted and targeted high-performance liquid chromatography coupled to mass spectrometry were performed on biological tissues and fluids. In our experimental conditions, a reversible BBBO was observed in the striatum without physical damage or a change in rodent weight and behavior. Cerebral, peri-cerebral, and peripheral metabolomes displayed specific and sequential metabolic kinetics. The blood serum metabolome was more impacted in terms of the number of perturbated metabolisms than in the CSF, the striatum, and the urine. In addition, perturbations of arginine and arginine-related metabolisms were detected in all matrices after BBBO, suggesting activation of vasomotor processes and bioenergetic supply. The exploration of the tryptophan metabolism revealed a transient vascular inflammation and a perturbation of serotoninergic neurotransmission in the striatum. For the first time, we characterized the metabolic signature following the acoustically-mediated BBBO within the striatum and its surrounding biological compartments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Presset, Bodard, Lefèvre, Millet, Oujagir, Dupuy, Iazourène, Bouakaz, Emond, Escoffre and Nadal-Desbarats.)

Introduction: The combination of microbubbles (MBs) and ultrasound (US) is an emerging method for the noninvasive and targeted enhancement of intratumor chemotherapeutic uptake. This method showed an increased local drug extravasation in tumor tissue while reducing the systemic adverse effects in various tumor models., Area Covered: We focused on preclinical and clinical studies investigating the therapeutic efficacy and safety of this technology for the treatment of colorectal, pancreatic, and liver cancers. We discussed the limitations of the current investigations and future perspectives., Expert Opinion: The therapeutic efficacy and the safety of delivery of standard chemotherapy regimen using MB-assisted US have been mainly demonstrated in subcutaneous models of digestive cancers. Although some clinical trials on pancreatic ductal carcinoma and hepatic metastases from various digestive cancers have shown promising results, successful evaluation of this method in terms of US settings, chemotherapeutic schemes, and MBs-related parameters will need to be addressed in more relevant preclinical models of digestive cancers, in small and large animals before fully and successfully translating this technology for clinic use. Ultimately, a clear evidence of the correlation between the enhanced intratumoral concentrations of therapeutics and the increased therapeutic response of tumors have to be provided in clinical trials.

Ultrasound neurostimulation (USNS) is being investigated as a treatment approach for neuropsychiatric and neurodegenerative disorders. Indeed, unlike the existing methods that use electric or magnetic stimulation, it offers the possibility to modulate brain activity in a noninvasive way, with good spatial specificity and a high penetration capacity. However, there is no consensus yet on ultrasound parameters and beam properties required for efficient neurostimulation. In this context, this preclinical study aimed to elucidate the effect of frequency, peak negative pressure (PNP), pulse duration (PD), and focal spot diameter, on the USNS efficiency. This was done by targeting the motor cortex (M1) of 70 healthy mice and analyzing the elicited motor responses (visually and with electromyography). Also, a further investigation was performed by assessing the corresponding neuronal activity, using c-Fos immunostaining. The results showed that the success rate, a metric that depicts USNS efficacy, increased with PNP in a sigmoidal way, reaching up to 100%. This was verified at different frequencies (0.5, 1, 1.5, and 2.25 MHz) and PDs (53.3, 160, and 320 ms, at 1.5 MHz fixed frequency). Moreover, it was shown that higher PNP values were required to achieve a constant USNS efficacy not only when frequency increased, but also when the focal spot diameter decreased, emphasizing a close link between these acoustic parameters and USNS efficacy. These findings were confirmed with immunohistochemistry (IHC), which showed a strong relationship between neural activation, the applied PNP, and the focal spot diameter.

Physical activity is increasingly recognized as a strategy able to improve cancer patient outcome, and its potential to enhance treatment response is promising, despite being unclear. In our study we used a preclinical model of prostate cancer to investigate whether voluntary wheel running (VWR) could improve tumor perfusion and enhance radiotherapy (RT) efficiency. Nude athymic mice were injected with PC-3 cancer cells and either remained inactive or were housed with running wheels. Apparent microbubble transport was enhanced with VWR, which we hypothesized could improve the RT response. When repeating the experiments and adding RT, however, we observed that VWR did not influence RT efficiency. These findings contrasted with previous results and prompted us to evaluate if the lack of effects observed on tumor growth could be attributable to the physical activity modality used. Using PC-3 and PPC-1 xenografts, we randomized mice to either inactive controls, VWR, or treadmill running (TR). In both models, TR (but not VWR) slowed down tumor growth, suggesting that the anti-cancer effects of physical activity are dependent on its modalities. Providing a better understanding of which activity type should be recommended to cancer patients thus appears essential to improve treatment outcomes.

Stroke is the second leading cause of death worldwide. Existing therapies present limitations, and other therapeutic alternatives are sought, such as sonothrombolysis with microbubbles (STL). The aim of this study was to evaluate the change induced by STL with or without recombinant tissue-type plasminogen activator (rtPA) on the acoustic and elastic properties of the blood clot by measuring its sound speed (SoS) and shear wave speed (SWS) with high frequency ultrasound and ultrafast imaging, respectively. An in-vitro setup was used and human blood clots were submitted to a combination of microbubbles and rtPA. The results demonstrate that STL induces a raise of SoS in the blood clot, specifically when combined with rtPA ( p < 0.05). Moreover, the combination of rtPA and STL induces a hardening of the clot in comparison to rtPA alone ( p < 0.05). This is the first assessment of acoustoelastic properties of blood clots during STL. The combination of rtPA and STL induce SoS and hardening of the clot, which is known to impair the penetration of thrombolytic drugs and their efficacy.

Background: Increasing evidence suggests that ultrasound (US) imaging may provide biomarkers and therapeutic options in mental disorders. We systematically reviewed the literature to provide a global overview of the possibilities of US for psychiatry., Methods: Original English language articles published between January 2000 and September 2019 were identified through databases searching and analyzed to summarize existing evidence according to PRISMA methodology., Results: A total of 81 articles were included. Various US techniques and markers have been used in mental disorders, including Transcranial Doppler and Intima-Media Thickness. Most of the studies have focused on characterizing the pathophysiology of mental disorders, especially vascular physiology. Studies on therapeutic applications are still scarce., Discussion: US imaging has proved to be useful in characterizing vascular impairment and structural and functional brain changes in mental disorders. Preliminary findings also suggest potential interests for therapeutic applications. Growing evidence suggests that US imaging could provide a non-invasive, portable and low-cost tool for pathophysiological characterization, prognostic assessment and therapeutic applications in mental disorders., Competing Interests: Declaration of competing interest WEH reports personal fees from EISAI, Japan, Janssen, USA, Lundbeck, Denmark, Otsuka, Japan, UCB, Belgium, Roche, Switzerland and Chugai, Japan. He received grants from the Fondation de France, France, and from the French National Hospital Program for Clinical Research (PHRC), unrelated to the submitted work. TD reports personal fees from Janssen, Lundbeck, Otsuka and Eisai. He received grants from the Fondation de l'Avenir, France, and from the French National Hospital Program for Clinical Research (PHRC), France, unrelated to the submitted work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microbubble-assisted ultrasound has emerged as a promising method for local drug delivery. Microbubbles are intravenously injected and locally activated by ultrasound, thus increasing the permeability of vascular endothelium for facilitating extravasation and drug uptake into the treated tissue. Thereby, endothelial cells are the first target of the effects of ultrasound-driven microbubbles. In this review, the in vitro and in vivo bioeffects of this method on endothelial cells are described and discussed, including aspects on the permeabilization of biologic barriers (endothelial cell plasma membranes and endothelial barriers), the restoration of their integrity, the molecular and cellular mechanisms involved in both these processes, and the resulting intracellular and intercellular consequences. Finally, the influence of the acoustic settings, microbubble parameters, treatment schedules and flow parameters on these bioeffects are also reviewed., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood-brain and blood-spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments., Competing Interests: Conflict of interest disclosure The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Laparoscopic surgery (LS) may lead to musculoskeletal disorders (MSDs) and an increase in physical and mental workloads to the surgeon. Robot-assisted surgery (RAS) should improve the ergonomy of the surgeon. This study assesses the experience influence in surgical ergonomics between LS and RAS., Methods: LS and RAS lasting more than 60 min of effective operative time were compared. During the surgical procedure, the physical discomfort was evaluated using the Borg scale. At the end, the mental workload was evaluated using the NASA-TLX index. After global analysis, the experienced and young surgeons were assessed., Results: 88 RAS and 82 LS were evaluated. During LS, the physical discomfort was significantly higher in all segments, and the pain increased significantly during the procedure in all segments compared to that evaluated in the RAS (p < 0.05). Forearms and the back were the most painful. The young surgeons did not display any improvement in the physical ergonomics of the RAS compared to the LS. Concerning the mental ergonomics, the overall workload and performance were significantly greater during the LS compared to the RAS (p < 0.05). For the young surgeons, the overall workload, the effort, the mental and the physical demands were greater during LS (p < 0.05). For the experienced surgeons, the physical demand was lower during the RAS compared to the LS (p < 0.05). However, the experienced surgeons expressed a feeling of greater performance after the LS (p < 0.01). RAS significantly reduces the onset of MSDs compared to LS, especially for the experienced surgeons., Conclusion: RAS significantly reduces the onset of MSDs compared to LS, especially for the experienced surgeons.

The poor diffusion of intravenous antibiotics in lung tissue makes nosocomial pneumonia challenging to treat, notably in critical patients under mechanical ventilation. The combination of ultrasound and microbubbles (USMB) is an emerging method for non-invasive and targeted enhancement of uptake of various drugs in several organs. This study aims to evaluate if USMB may increase amikacin concentration in condensed lung tissues in a mechanically ventilated rabbit model. When applied 60 or 160 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the condensed lung tissue by 1.33 ( p = 0.025) or 1.56-fold ( p = 0.028) respectively. When applied 70 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the muscle tissue by 2.52 ( p = 0.025). In conclusion, this study demonstrates that USMB is a promising method for the targeted delivery of amikacin in mechanically ventilated condensed lung, thus opening new therapeutic fields against lung infections., (Copyright © 2020 Espitalier, Darrouzain, Escoffre, Ternant, Piver, Bouakaz and Remerand.)

A combination of microbubbles (MBs) and ultrasound (US) is an emerging method for noninvasive and targeted enhancement of anti-cancer drug uptake. This method showed an increase local drug extravasation in tumor tissue while reducing the systemic adverse effects in various tumor models. The present study aims to evaluate the effectiveness of this approach for Nab-paclitaxel delivery in a pancreatic tumor model. US and MBs of different types in combination with Nab-paclitaxel showed a loss in cell viability of pancreatic cancer cells in comparison with Nab-paclitaxel treatment alone in in vitro scenario. The in vivo data revealed that US and MBs in combination with Nab-paclitaxel induced a significant decrease in the tumor volume in a subcutaneous pancreatic adenocarcinoma mouse model in comparison to tumors treated with Nab-paclitaxel alone. The postmortem anatomopathological analyses of tumor tissues partially confirmed these results. In conclusion, this study demonstrates that MB-assisted US is a relevant technology to increase the therapeutic effectiveness of Nab-paclitaxel in a pancreatic cancer model.

Microbubble-assisted ultrasound has emerged as a promising method for the delivery of low-molecular-weight chemotherapeutic molecules, nucleic acids, therapeutic peptides, and antibodies in vitro and in vivo. Its clinical applications are under investigation for local delivery drug in oncology and neurology. However, the biophysical mechanisms supporting the acoustically mediated membrane permeabilization are not fully established. This review describes the present state of the investigations concerning the acoustically mediated stimuli (i.e., mechanical, chemical, and thermal stimuli) as well as the molecular and cellular actors (i.e., membrane pores and endocytosis) involved in the reversible membrane permeabilization process. The different hypotheses, which were proposed to give a biophysical description of the membrane permeabilization, are critically discussed.

Liver fibrosis is the common result of chronic liver disease. Diagnosis and grading liver fibrosis for patient management is mainly based on blood tests and hepatic puncture-biopsy, which is particularly invasive. Quantitative ultrasound (QUS) techniques provide insight into tissue microstructure and are based on the frequency-based analysis of the signals from biologic tissues. This study aims to quantify how spectral-based QUS parameters change with fibrosis grade. The changes in QUS parameters of healthy and fibrotic rabbit liver samples were investigated and were compared with the changes in liver stiffness, using shear wave elastography. Overall, the acoustic concentration was found to decrease with increasing fibrosis grade, and the effective scatterer size was found to be higher in fibrotic livers when compared with normal liver. The result of this study indicates that the combination of three QUS parameters (stiffness, effective scatterer size and acoustic concentration) provides the best classification performance, especially for classifying healthy and fibrotic livers., (Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

In solid tumors, rapid local intravascular release of anticancer agents, e.g., doxorubicin (DOX), from thermosensitive liposomes (TSLs) can be an option to overcome poor extravasation of drug nanocarriers. The driving force of DOX penetration is the drug concentration gradient between the vascular compartment and the tumor interstitium. In this feasibility study, we used fibered confocal fluorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumor after its intravascular release from TSLs, Thermodox®. Cell uptake kinetics of the released DOX was quantified, along with an in-depth assessment of released-DOX penetration using an evolution model. A subcutaneous rat R1 rhabdomyosarcoma xenograft was used. The rodent was positioned in a setup including a water bath, and FCFM identification of functional vessels in the tumor tissue was applied based on AngioSense. The tumor-bearing leg was immersed in the 43°C water for preheating, and TSLs were injected intravenously. Real-time monitoring of intratumoral (i.t.) DOX penetration could be performed, and it showed the progressing DOX wave front via its native fluorescence, labeling successively all cell nuclei. Cell uptake rates (1/k) of 3 minutes were found ( n =241  cells), and a released-DOX penetration in the range of 2500  µ m 2 ·s -1 was found in the tumor extravascular space. This study also showed that not all vessels, identified as functional based on AngioSense, gave rise to local DOX penetration.

The axial resolution of an ultrasound imaging system is inversely proportional to the bandwidth of the emitted signal. When conventional pulsing (CP) is used, the impulse response of the transducer and the excitation signal determine together the shape of the emitted pulse and its bandwidth. A way to increase the ultrasound image resolution is to increase the transducer's limited passband. The resolution enhancement compression (REC) is a coding technique that boosts the signal energy in the transition frequency bands, where the energy transduction of the ultrasound probe is less efficient. Consequently, image quality metrics including axial resolution, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) can be improved. In this paper, the objective is to combine REC with coherent plane-wave compounding (CPWC) in order to achieve better image quality at an ultrafast acquisition rate. Promising results are obtained from both wire and cyst phantoms using an excitation signal designed to provide a 54% increase in bandwidth over the one obtained with a broadband pulse excitation at -6 dB. The experimental bandwidth measured from the backscattered echoes was improved by 49% for the wire phantom, when using the CPWC-REC technique compared to CPWC-CP. Furthermore, the axial resolution as derived from the modulation transfer function of the envelope of the wire target was enhanced by 29%. The CNR and SNR were improved up to 9 and up to 4 dB, respectively, in the cyst phantom. These results reveal that CPWC-REC is able to achieve higher spatial resolution, compared to CPWC-CP, with better SNR and CNR. Moreover, experimental results show that an effective implementation on a research scanner of REC using plane-wave imaging is possible. Consistent in vivo acquisition results on rabbit are presented and discussed.

Background: Microbubbles (MBs) combined with ultrasound sonothrombolysis (STL) appears to be an alternative therapeutic strategy for acute ischemic stroke (IS), but clinical results remain controversial., Objective: The aim of this systematic review is to identify the parameters tested; to assess evidence on the safety and efficacy on preclinical data on STL; and to assess the validity and publication bias., Methods: Pubmed® and Web of ScienceTM databases were systematically searched from January 1995 to April 2017 in French and English. We included studies evaluating STL on animal stroke model. This systematic review was conducted in accordance with the PRISMA guidelines. Data were extracted following a pre-defined schedule by two of the authors. The CAMARADES criteria were used for quality assessment. A narrative synthesis was conducted., Results: Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited., Limitations: Quality assessment of the studies reviewed revealed a number of biases., Conclusion: Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options., Systematic Review Registration: http://syrf.org.uk/protocols/.

Contrast-enhanced ultrasound (CEUS) is a non-invasive imaging technique extensively used for blood perfusion imaging of various organs. This modality is based on the acoustic detection of gas-filled microbubble contrast agents used as intravascular flow tracers. Recent efforts aim at quantifying parameters related to the enhancement in the vascular compartment using time-intensity curve (TIC), and at using these latter as indicators for several pathological conditions. However, this quantification is mainly hampered by two reasons: first, the quantification intrinsically solely relies on temporal intensity variation, the explicit spatial transport of the contrast agent being left out. Second, the exact relationship between the acquired US-signal and the local microbubble concentration is hardly accessible. This paper introduces the use of a fluid dynamic model for the analysis of dynamic CEUS (DCEUS), in order to circumvent the two above-mentioned limitations. A new kinetic analysis is proposed in order to quantify the velocity amplitude of the bolus arrival. The efficiency of proposed methodology is evaluated both in-vitro, for the quantitative estimation of microbubble flow rates, and in-vivo, for the classification of placental insufficiency (control versus ligature) of pregnant rats from DCEUS. Besides, for the in-vivo experimental setup, we demonstrated that the proposed approach outperforms the performance of existing TIC-based methods.

Introduction: Recent developments in ultrasound imaging and ultrasound contrast agents (UCAs) improved diagnostic confidence in echography and set into motion their combined use as a tool for drug delivery and therapeutic monitoring. Non-invasive, precise and targeted delivery of drug molecules to pathological tissues by employing different mechanisms of drug release is becoming feasible. Areas covered: We sought to describe: the nature and features of UCAs; outline current contrast-specific imaging modes; before describing a variety of strategies for using ultrasound and microbubbles as a drug delivery system. Our expert opinion focusses on results and prospects of using ultrasound and microbubbles as a dual modality for drug delivery and therapeutic monitoring. Expert opinion: Today, ultrasound and microbubbles present a realistic prospect as drug delivery tools that have been demonstrated in a variety of animal models and clinical indications. Besides delivering drugs, ultrasound and microbubbles have demonstrated added value through therapeutic monitoring and assessment. Successful evaluation of the sonoporation mechanism(s), ultrasound parameters, drug type and dose will need to be addressed before translating this technology for clinic use. Ultimately, the development of a strategy for monitoring targeted delivery and its implementation in clinical practice would advance therapeutic treatment to a new qualitative level.

Thrombosis is a major cause of several diseases, i.e. myocardial infarction, cerebral stroke and pulmonary embolism. Thrombolytic therapies are required to induce fast and efficient recanalization of occluded vessels. To evaluate the in vitro efficacy of these thrombolytic strategies, measuring clot dissolution is essential. This study aimed to evaluate and validate high resolution ultrasound as a tool to assess the exact volume of clots in 3D and in real time during in vitro thrombolytic drug testing. This new method was validated by measuring the effects of concentration range of recombinant tissue type plasminogen activator on a blood clot during complete occlusion or 70% stenosis of a vessel. This study shows that high resolution ultrasound imaging allows for a real-time assessment of the 3D volume of a blood clot with negligible inter- and intra-operator variabilities. The conclusions drawn from this study demonstrate the promising potential of high resolution ultrasound imaging for the in vitro assessment of new thrombolytic drugs.

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Purpose: This preclinical study aimed to evaluate placental oxygenation in pregnant rats by real-time photoacoustic (PA) imaging on different days of gestation and to specify variations in placental oxygen saturation under conditions of maternal hypoxia and hyperoxygenation., Material and Methods: Placentas of fifteen Sprague-Dawley rats were examined on days 14, 17, and 20 of pregnancy with a PA imaging system coupled to high-resolution ultrasound imaging. Pregnant rats were successively exposed to hyperoxygenated and hypoxic conditions by changing the oxygen concentration in inhaled gas. Tissue oxygen saturation was quantitatively analyzed by real-time PA imaging in the skin and 3 regions of the placenta. All procedures were performed in accordance with applicable ethical guidelines and approved by the animal care committee., Results: Maternal hypoxia was associated with significantly greater decrease in blood oxygen saturation (ΔO2 Saturation) in the skin (70.74% ±7.65) than in the mesometrial triangle (32.66% ±5.75) or other placental areas (labyrinth: 18.58% ± 6.61; basal zone: 13.13% ±5.72) on different days of pregnancy (P<0.001). ΔO2 Saturation did not differ significantly between the labyrinth, the basal zone, and the decidua. After the period of hypoxia, maternal hyperoxygenation led to a significant rise in oxygen saturation, which returned to its initial values in the different placental regions (P<0.001)., Conclusions: PA imaging enables the variation of blood oxygen saturation to be monitored in the placenta during maternal hypoxia or hyperoxygenation. This first preclinical study suggests that the placenta plays an important role in protecting the fetus against maternal hypoxia., Competing Interests: The authors have declared that no competing interests exist.

Bubble-assisted ultrasound is a versatile technology with great potential in immunotherapy and vaccination. This technology involves the exposure of immune cells (i.e., dendritic cells, lymphocytes) in-vitro or diseased tissues (i.e., brain, tumor) in-vivo to ultrasound treatment with gas bubbles. Bubble destruction leads to physical forces that induce the direct delivery of weakly permeant immuno-stimulatory molecules either into the cytoplasm of immune cells, or through the endothelial barrier of diseased tissues. Hence, therapeutic antibodies (i.e., antibody-based immunotherapy) and cytokine-encoding nucleic acids (i.e., cytokine gene therapy) can be successfully delivered into diseased tissues, thus improving immune responses. In addition, protein antigens, as well as antigen-encoding nucleic acids (pDNA, mRNA), can be delivered into dendritic cells (i.e., dendritic cell-based vaccines), thus leading to a long-lasting prophylactic or therapeutic immunization. This chapter focuses on the state-of-the-art of bubble-assisted ultrasound in the field of immunotherapy and vaccination.

Sonoporation is a physical method that has been successfully used to deliver drugs into living cells both in vitro and in vivo for experimental and therapeutic purposes. Despite numerous studies on this topic, often reporting successful outcomes, very little is known about the mechanisms involved in the hypothesized membrane permeabilization processes. In this study, electron microscopy was used to investigate the ultra-structural modifications of cell membranes, induced by sonoporation. Here, we demonstrate that sonoporation in the presence of microbubbles induces the formation of a significant number of transient and permeant structures at the membrane level. These structures were transient with a half-life of 10 min and had a heterogeneous size distribution ranging from a few nanometers to 150 nm. We demonstrated that the number and the size of these structures were positively correlated with the enhanced intracellular uptake of small molecules. In addition, we showed that these structures were associated with caveolae-dependent endocytosis for two thirds of the recorded events, with the remaining one third related to non-specific routes such as membrane disruptions as well as caveolae-independent endocytosis. In conclusion, our observations provide direct evidences of the involvement of caveolae-endocytosis in cell membrane permeabilization to small molecules after sonoporation.

The combination of microbubbles and ultrasound has emerged as a promising method for local drug delivery. Microbubbles can be locally activated by a targeted ultrasound beam, which can result in several bio-effects. For drug delivery, microbubble-assisted ultrasound is used to increase vascular- and plasma membrane permeability for facilitating drug extravasation and the cellular uptake of drugs in the treated region, respectively. In the case of drug-loaded microbubbles, these two mechanisms can be combined with local release of the drug following destruction of the microbubble. The use of microbubble-assisted ultrasound to deliver chemotherapeutic agents is also referred to as sonochemotherapy. In this review, the basic principles of sonochemotherapy are discussed, including aspects such as the type of (drug-loaded) microbubbles used, the routes of administration used in vivo, ultrasound devices and parameters, treatment schedules and safety issues. Finally, the clinical translation of sonochemotherapy is discussed, including the first clinical study using sonochemotherapy.

Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue(®) MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

Focused ultrasound (FUS) is a versatile technology for non-invasive thermal therapies in oncology. Indeed, this technology has great potential for local heat-mediated drug delivery from thermosensitive liposomes (TSLs), thus improving therapeutic efficacy and reducing toxicity profiles. In the present study we evaluated the influence of FUS parameters on the release of calcein from TSLs used to model a hydrophilic drug. Quantitative calcein release from TSLs (DPPC/CHOL/DSPE-PEG2000: 90/5/5) and non-thermosensitive liposomes (NTSLs) (DPPC/CHOL/DSPE-PEG2000: 65/30/5) was measured by spectrofluorimetry after both water bath and FUS-induced in vitro heating. The heating of TSLs at 42 °C in a water bath resulted in a maximum calcein release of 45%. No additional calcein release was observed at temperatures above 42 °C. A similar percentage of calcein release was achieved when TSLs were exposed to 1 MHz sinusoidal waves at peak negative pressure of 1.5 MPa, 40% duty cycle, for 10 min (i.e. above 42 °C). No release was detected when NTSLs were heated in a water bath. For both TSLs and NTSLs, the calcein release was increased by more than 10% for acoustic pressures ranging from 1.5 MPa to 2 MPa. This additional release was attributed to the mechanical stress generated by FUS, which was sufficient to disrupt the liposomal membrane. Furthermore, analysis of cryo-TEM images showed a significant decrease in liposome size (14%) induced by the thermal effect, whereas the liposome diameter remained unaffected by the FUS-triggered non-thermal effects.