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Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery

  • Klazina Kooiman
    Correspondence
    Address correspondence to: Klazina Kooiman, Office Ee2302, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
    Affiliations
    Department of Biomedical Engineering, Thoraxcenter, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands
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  • Silke Roovers
    Affiliations
    Ghent Research Group on Nanomedicines, Lab for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
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  • Simone A.G. Langeveld
    Affiliations
    Department of Biomedical Engineering, Thoraxcenter, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands
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  • Robert T. Kleven
    Affiliations
    Department of Biomedical Engineering, College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, USA
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  • Heleen Dewitte
    Affiliations
    Ghent Research Group on Nanomedicines, Lab for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ghent, Belgium

    Laboratory for Molecular and Cellular Therapy, Medical School of the Vrije Universiteit Brussel, Jette, Belgium

    Cancer Research Institute Ghent (CRIG), Ghent University Hospital, Ghent University, Ghent, Belgium
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  • Meaghan A. O'Reilly
    Affiliations
    Physical Sciences Platform, Sunnybrook Research Institute, Toronto, Ontario, Canada

    Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
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  • Jean-Michel Escoffre
    Affiliations
    UMR 1253, iBrain, Université de Tours, Inserm, Tours, France
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  • Ayache Bouakaz
    Affiliations
    UMR 1253, iBrain, Université de Tours, Inserm, Tours, France
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  • Martin D. Verweij
    Affiliations
    Department of Biomedical Engineering, Thoraxcenter, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands

    Laboratory of Acoustical Wavefield Imaging, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands
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  • Kullervo Hynynen
    Affiliations
    Physical Sciences Platform, Sunnybrook Research Institute, Toronto, Ontario, Canada

    Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada
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  • Ine Lentacker
    Affiliations
    Ghent Research Group on Nanomedicines, Lab for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, Ghent, Belgium

    Cancer Research Institute Ghent (CRIG), Ghent University Hospital, Ghent University, Ghent, Belgium
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  • Eleanor Stride
    Affiliations
    Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom
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  • Christy K. Holland
    Affiliations
    Department of Biomedical Engineering, College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, USA

    Department of Internal Medicine, Division of Cardiovascular Health and Disease, University of Cincinnati, Cincinnati, OH, USA
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Open AccessPublished:March 09, 2020DOI:https://doi.org/10.1016/j.ultrasmedbio.2020.01.002

      Abstract

      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.

      Key Words

      Introduction

      Around the start of the European Symposium on Ultrasound Contrast Agents, ultrasound-responsive cavitation nuclei were reported to have therapeutic potential. Thrombolysis was reported to be accelerated in vitro (
      • Tachibana K.
      • Tachibana S.
      Albumin microbubble echo-contrast material as an enhancer for ultrasound accelerated thrombolysis.
      ), and cultured cells were transfected with plasmid DNA (
      • Bao S.
      • Thrall B.D.
      • Miller D.L.
      Transfection of a reporter plasmid into cultured cells by sonoporation in vitro.
      ). Since then, many research groups have investigated the use of cavitation nuclei for multiple forms of therapy, including tissue ablation and drug and gene delivery. In the early years, the most widely investigated cavitation nuclei were gas microbubbles, ∼1–10 µm in diameter and coated with a stabilizing shell, whereas today both solid and liquid nuclei, which can be as small as a few hundred nanometers, are also being investigated. Drugs can be co-administered with the cavitation nuclei or loaded in or on them (
      • Lentacker I.
      • De Smedt S.C.
      • Sanders N.N.
      Drug loaded microbubble design for ultrasound triggered delivery.
      ;
      • Kooiman K.
      • Vos H.J.
      • Versluis M.
      • de Jong N.
      Acoustic behavior of microbubbles and implications for drug delivery.
      ). The diseases that can be treated with ultrasound-responsive cavitation nuclei include but are not limited to cardiovascular disease and cancer (
      • Sutton J.T.
      • Haworth K.J.
      • Pyne-Geithman G.
      • Holland C.K.
      Ultrasound-mediated drug delivery for cardiovascular disease.
      ;
      • Paefgen V.
      • Doleschel D.
      • Kiessling F.
      Evolution of contrast agents for ultrasound imaging and ultrasound-mediated drug delivery.
      ), the current leading causes of death worldwide according to the World Health Organization (
      • Nowbar A.N.
      • Gitto M.
      • Howard J.P.
      • Francis D.P.
      • Al-Lamee R.
      Mortality From ischemic heart disease: Analysis of data from the World Health Organization and coronary artery disease risk factors from NCD risk factor collaboration.
      ). This review focuses on the latest insights into cavitation nuclei for therapy and drug delivery from the physical and biological mechanisms of bubble–cell interaction to pre-clinical (both in vitro and in vivo) and clinical (time span: 2014-2019) studies, with particular emphasis on the key clinical applications. The applications covered in this review are the treatment of tumors, immunotherapy, blood–brain barrier (BBB) and blood–spinal cord barrier, dissolution of clots, cardiovascular drug delivery and treatment of bacterial infections.

      Cavitation nuclei for therapy

      The most widely used cavitation nuclei are phospholipid-coated microbubbles with a gas core. For the 128 pre-clinical studies included in the treatment sections of this review, the commercially available and clinically approved Definity (Luminity in Europe; octafluoropropane gas core, phospholipid coating) (
      Definity
      Silver Spring.
      ;
      • Nolsøe C.P.
      • Lorentzen T.
      International guidelines for contrast-enhanced ultrasonography: Ultrasound imaging in the new millennium.
      ) microbubbles were the most frequently used (in 22 studies). Definity was used for studies on all applications discussed here, mostly for opening the BBB (12 studies). SonoVue (Lumason in the United States) is commercially available and clinically approved as well (sulfur hexafluoride gas core, phospholipid coating) (
      Lumason
      Silver Spring.
      ;
      • Nolsøe C.P.
      • Lorentzen T.
      International guidelines for contrast-enhanced ultrasonography: Ultrasound imaging in the new millennium.
      ) and was used in a total of 14 studies for treatment of non-brain tumors (e.g.,
      • Xing L.
      • Shi Q.
      • Zheng K.
      • Shen M.
      • Ma J.
      • Li F.
      • Liu Y.
      • Lin L.
      • Tu W.
      • Duan Y.
      • Du L.
      Ultrasound-mediated microbubble destruction (UMMD) facilitates the delivery of CA19-9 targeted and paclitaxel loaded mPEG-PLGA-PLL nanoparticles in pancreatic cancer.
      ), BBB opening (e.g.,
      • Goutal S.
      • Gerstenmayer M.
      • Auvity S.
      • Caillé F.
      • Mériaux S.
      • Buvat I.
      • Larrat B.
      • Tournier N.
      Physical blood-brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib.
      ) and sonobactericide (e.g.,
      • Hu J.
      • Zhang Jr, N.
      • Li L.
      • Zhang Sr, N.
      • Ma Y.
      • Zhao C.
      • Wu Q.
      • Li Y.
      • He N.
      • Wang X.
      The synergistic bactericidal effect of vancomycin on UTMD treated biofilm involves damage to bacterial cells and enhancement of metabolic activities.
      ). Other commercially available microbubbles were used that are not clinically approved, such as BR38 (
      • Schneider M.
      • Anantharam B.
      • Arditi M.
      • Bokor D.
      • Broillet A.
      • Bussat P.
      • Fouillet X.
      • Frinking P.
      • Tardy I.
      • Terrettaz J.
      • Senior R.
      • Tranquart F.
      BR38, a new ultrasound blood pool agent.
      ) in the study by
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      and MicroMarker (
      VisualSonics
      PN11691—Vevo MicroMarker Non-Targeted Contrast Agent Kit: Protocol and Information Booklet Rev. 1.4.
      ) in the study by
      • Theek B.
      • Baues M.
      • Ojha T.
      • Mockel D.
      • Veettil S.K.
      • Steitz J.
      • van Bloois L.
      • Storm G.
      • Kiessling F.
      • Lammers T.
      Sonoporation enhances liposome accumulation and penetration in tumors with low EPR.
      . Custom-made microbubbles are as diverse as their applications, with special characteristics tailored to enhance different therapeutic strategies. Different types of gasses were used as the core such as air (e.g.,
      • Eggen S.
      • Fagerland S.M.
      • Mørch Ý.
      • Hansen R.
      • Søvik K.
      • Berg S.
      • Furu H.
      • Bøhn A.D.
      • Lilledahl M.B.
      • Angelsen A.
      • Angelsen B.
      • de Lange Davies C.
      Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.
      ), nitrogen (e.g.,
      • Dixon A.J.
      • Li J.
      • Rickel J.M.R.
      • Klibanov A.L.
      • Zuo Z.Y.
      • Hossack J.A.
      Efficacy of sonothrombolysis using microbubbles produced by a catheter-based microfluidic device in a rat model of ischemic stroke.
      ), oxygen (e.g.,
      • Fix S.M.
      • Papadopoulou V.
      • Velds H.
      • Kasoji S.K.
      • Rivera J.N.
      • Borden M.A.
      • Chang S.
      • Dayton P.A.
      Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.
      ), octafluoropropane (e.g.,
      • Pandit R.
      • Leinenga G.
      • Götz J.
      Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.
      ), perfluorobutane (e.g.,
      • Dewitte H.
      • Vanderperren K.
      • Haers H.
      • Stock E.
      • Duchateau L.
      • Hesta M.
      • Saunders J.H.
      • De Smedt S.C.
      • Lentacker I.
      • De S.C.
      Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: Implications for drug delivery.
      ), sulfur hexafluoride (
      • Bae Y.J.
      • Yoon Y.I.
      • Yoon T.J.
      • Lee H.J.
      Ultrasound-guided delivery of siRNA and a chemotherapeutic drug by using microbubble complexes: In vitro and in vivo evaluations in a prostate cancer model.
      ;
      • Horsley H.
      • Owen J.
      • Browning R.
      • Carugo D.
      • Malone-Lee J.
      • Stride E.
      • Rohn J.L.
      Ultrasound-activated microbubbles as a novel intracellular drug delivery system for urinary tract infection.
      ) or a mixture of gases such as nitric oxide and octafluoropropane (
      • Sutton J.T.
      • Raymond J.L.
      • Verleye M.C.
      • Pyne-Geithman G.J.
      • Holland C.K.
      Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue.
      ) or sulfur hexafluoride and oxygen (
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ). While fluorinated gases improve the stability of phospholipid-coated microbubbles (
      • Rossi S.
      • Szíjjártó C.
      • Gerber F.
      • Waton G.
      • Krafft M.P.
      Fluorous materials in microbubble engineering science and technology—Design and development of new bubble preparation and sizing technologies.
      ), other gases can be loaded for therapeutic applications, such as oxygen for treatment of tumors (
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ;
      • Fix S.M.
      • Papadopoulou V.
      • Velds H.
      • Kasoji S.K.
      • Rivera J.N.
      • Borden M.A.
      • Chang S.
      • Dayton P.A.
      Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.
      ;
      • Nesbitt H.
      • Sheng Y.
      • Kamila S.
      • Logan K.
      • Thomas K.
      • Callan B.
      • Taylor M.A.
      • Love M.
      • O'Rourke D.
      • Kelly P.
      • Beguin E.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.
      ) and nitric oxide (
      • Kim H.
      • Britton G.L.
      • Peng T.
      • Holland C.K.
      • McPherson D.D.
      • Huang S.L.
      Nitric oxide-loaded echogenic liposomes for treatment of vasospasm following subarachnoid hemorrhage.
      ;
      • Sutton J.T.
      • Raymond J.L.
      • Verleye M.C.
      • Pyne-Geithman G.J.
      • Holland C.K.
      Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue.
      ) and hydrogen gas (
      • He Y.
      • Zhang B.
      • Chen Y.
      • Jin Q.
      • Wu J.
      • Yan F.
      • Zheng H.
      Image-guided hydrogen gas delivery for protection from myocardial ischemia–reperfusion injury via microbubbles.
      ) for treatment of cardiovascular disease. The main phospholipid component of custom-made microbubbles is usually a phosphatidylcholine such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), used in 13 studies (e.g.,
      • Dewitte H.
      • Vanderperren K.
      • Haers H.
      • Stock E.
      • Duchateau L.
      • Hesta M.
      • Saunders J.H.
      • De Smedt S.C.
      • Lentacker I.
      • De S.C.
      Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: Implications for drug delivery.
      ;
      • Bae Y.J.
      • Yoon Y.I.
      • Yoon T.J.
      • Lee H.J.
      Ultrasound-guided delivery of siRNA and a chemotherapeutic drug by using microbubble complexes: In vitro and in vivo evaluations in a prostate cancer model.
      ;
      • Chen S.
      • Chen J.
      • Meng X.L.
      • Shen J.S.
      • Huang J.
      • Huang P.
      • Pu Z.
      • McNeill N.H.
      • Grayburn P.A.
      ANGPTL8 reverses established adriamycin cardiomyopathy by stimulating adult cardiac progenitor cells.
      ;
      • Fu Y.Y.
      • Zhang L.
      • Yang Y.
      • Liu C.W.
      • He Y.N.
      • Li P.
      • Yu X.
      Synergistic antibacterial effect of ultrasound microbubbles combined with chitosan-modified polymyxin B-loaded liposomes on biofilm-producing Acinetobacter baumannii.
      ), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), used in 18 studies (e.g.,
      • Kilroy J.P.
      • Klibanov A.L.
      • Wamhoff B.R.
      • Bowles D.K.
      • Hossack J.A.
      Localized in vivo model drug delivery with intravascular ultrasound and microbubbles.
      ;
      • Bioley G.
      • Lassus A.
      • Terrettaz J.
      • Tranquart F.
      • Corthesy B.
      Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria.
      ;
      • Dong Y.
      • Xu Y.
      • Li P.
      • Wang C.
      • Cao Y.
      • Yu J.
      Antibiofilm effect of ultrasound combined with microbubbles against Staphylococcus epidermidis biofilm.
      ;
      • Goyal A.
      • Yu F.T.H.
      • Tenwalde M.G.
      • Chen X.C.
      • Althouse A.
      • Villanueva F.S.
      • Pacella J.J.
      Inertial cavitation ultrasound with microbubbles improves reperfusion efficacy when combined with tissue plasminogen activator in an in vitro model of microvascular obstruction.
      ;
      • Pandit R.
      • Leinenga G.
      • Götz J.
      Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.
      ). These phospholipids are popular because they are also the main components in Definity (
      Definity
      Silver Spring.
      ) and SonoVue/Lumason (
      Lumason
      Silver Spring.
      ), respectively. Another key component of the microbubble coating is a polyethylene glycol (PEG)ylated emulsifier such as polyoxyethylene (40) stearate (PEG40-stearate; e.g.,
      • Kilroy J.P.
      • Klibanov A.L.
      • Wamhoff B.R.
      • Bowles D.K.
      • Hossack J.A.
      Localized in vivo model drug delivery with intravascular ultrasound and microbubbles.
      ) or the most frequently used 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol) (DSPE–PEG2000; e.g.,
      • Belcik J.T.
      • Davidson B.P.
      • Xie A.
      • Wu M.D.
      • Yadava M.
      • Qi Y.
      • Liang S.
      • Chon C.R.
      • Ammi A.Y.
      • Field J.
      • Harmann L.
      • Chilian W.M.
      • Linden J.
      • Lindner J.R.
      Augmentation of muscle blood flow by ultrasound cavitation is mediated by ATP and purinergic signaling.
      ), which is added to inhibit coalescence and to increase the in vivo half-life (
      • Ferrara K.W.
      • Borden M.A.
      • Zhang H.
      Lipid-shelled vehicles: Engineering for ultrasound molecular imaging and drug delivery.
      ). In general, two methods are used to produce custom-made microbubbles: mechanical agitation (e.g.,
      • Ho Y.J.
      • Wang T.C.
      • Fan C.H.
      • Yeh C.K.
      Spatially uniform tumor treatment and drug penetration by regulating ultrasound with microbubbles.
      ) and probe sonication (e.g.,
      • Belcik J.T.
      • Mott B.H.
      • Xie A.
      • Zhao Y.
      • Kim S.
      • Lindner N.J.
      • Ammi A.
      • Linden J.M.
      • Lindner J.R.
      Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.
      ). Both methods produce a population of microbubbles that is polydisperse in size. Monodispersed microbubbles produced by microfluidics have recently been developed, and are starting to gain attention for pre-clinical therapeutic studies.
      • Dixon A.J.
      • Li J.
      • Rickel J.M.R.
      • Klibanov A.L.
      • Zuo Z.Y.
      • Hossack J.A.
      Efficacy of sonothrombolysis using microbubbles produced by a catheter-based microfluidic device in a rat model of ischemic stroke.
      used monodisperse microbubbles to treat ischemic stroke.
      Various therapeutic applications have inspired the development of novel cavitation nuclei, which is discussed in depth in the companion review by
      • Stride E.
      • Lajoinie G.
      • Borden M.
      • Versluis M.
      • Cherkaoui S.
      • Bettinger T.
      • Segers T.
      Microbubble agents: New directions.
      . To improve drug delivery, therapeutics can be either co-administered with or loaded onto the microbubbles. One strategy for loading is to create microbubbles stabilized by drug-containing polymeric nanoparticles around a gas core (
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ). Another strategy is to attach therapeutic molecules or liposomes to the outside of microbubbles, for example, by biotin–avidin coupling (
      • Dewitte H.
      • Vanderperren K.
      • Haers H.
      • Stock E.
      • Duchateau L.
      • Hesta M.
      • Saunders J.H.
      • De Smedt S.C.
      • Lentacker I.
      • De S.C.
      Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: Implications for drug delivery.
      ;
      • McEwan C.
      • Kamila S.
      • Owen J.
      • Nesbitt H.
      • Callan B.
      • Borden M.
      • Nomikou N.
      • Hamoudi R.A.
      • Taylor M.A.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Combined sonodynamic and antimetabolite therapy for the improved treatment of pancreatic cancer using oxygen loaded microbubbles as a delivery vehicle.
      ;
      • Nesbitt H.
      • Sheng Y.
      • Kamila S.
      • Logan K.
      • Thomas K.
      • Callan B.
      • Taylor M.A.
      • Love M.
      • O'Rourke D.
      • Kelly P.
      • Beguin E.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.
      ). Echogenic liposomes can be loaded with different therapeutics or gases and have been studied for vascular drug delivery (
      • Sutton J.T.
      • Raymond J.L.
      • Verleye M.C.
      • Pyne-Geithman G.J.
      • Holland C.K.
      Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue.
      ), treatment of tumors (
      • Choi J.J.
      • Carlisle R.C.
      • Coviello C.
      • Seymour L.
      • Coussios C.-C.
      Non-invasive and real-time passive acoustic mapping of ultrasound-mediated drug delivery.
      ) and sonothrombolysis (
      • Shekhar H.
      • Bader K.B.
      • Huang S.W.
      • Peng T.
      • Huang S.L.
      • McPherson D.D.
      • Holland C.K.
      In vitro thrombolytic efficacy of echogenic liposomes loaded with tissue plasminogen activator and octafluoropropane gas.
      ). Acoustic Cluster Therapy (ACT) combines Sonazoid microbubbles with droplets that can be loaded with therapeutics for treatment of tumors (
      • Kotopoulis S.
      • Stigen E.
      • Popa M.
      • Safont M.M.
      • Healey A.
      • Kvåle S.
      • Sontum P.
      • Gjertsen B.T.
      • Gilja O.H.
      • McCormack E.
      Sonoporation with Acoustic Cluster Therapy (ACT) induces transient tumour volume reduction in a subcutaneous xenograft model of pancreatic ductal adenocarcinoma.
      ). The cationic microbubbles utilized in the treatment sections of this review were used mostly for vascular drug delivery, with genetic material loaded on the microbubble surface by charge coupling (e.g.,
      • Cao W.J.
      • Rosenblat J.D.
      • Roth N.C.
      • Kuliszewski M.A.
      • Matkar P.N.
      • Rudenko D.
      • Liao C.
      • Lee P.J.
      • Leong-Poi H.
      Therapeutic angiogenesis by ultrasound-mediated microRNA-126-3p delivery.
      ). Besides phospholipids and nanoparticles, microbubbles can also be coated with denatured proteins such as albumin. Optison (
      Optison
      Silver Spring.
      ) is a commercially available and clinically approved ultrasound contrast agent that is coated with human albumin and used in studies on treatment of non-brain tumors (
      • Xiao N.
      • Liu J.
      • Liao L.
      • Sun J.
      • Jin W.
      • Shu X.
      Ultrasound combined with microbubbles increase the delivery of doxorubicin by reducing the interstitial fluid pressure.
      ), BBB opening (
      • Kovacs Z.I.
      • Kim S.
      • Jikaria N.
      • Qureshi F.
      • Milo B.
      • Lewis B.K.
      • Bresler M.
      • Burks S.R.
      • Frank J.A.
      Disrupting the blood-brain barrier by focused ultrasound induces sterile inflammation.
      ;
      • Payne A.H.
      • Hawryluk G.W.
      • Anzai Y.
      • Odéen H.
      • Ostlie M.A.
      • Reichert E.C.
      • Stump A.J.
      • Minoshima S.
      • Cross D.J.
      Magnetic resonance imaging-guided focused ultrasound to increase localized blood-spinal cord barrier permeability.
      ) and immunotherapy (
      • Sta Maria N.S.
      • Barnes S.R.
      • Weist M.R.
      • Colcher D.
      • Raubitschek A.A.
      • Jacobs R.E.
      Low dose focused ultrasound induces enhanced tumor accumulation of natural killer cells.
      ). Nano-sized particles cited in this review have been used as cavitation nuclei for treatment of tumors, such as nanodroplets (e.g.,
      • Cao Y.
      • Chen Y.
      • Yu T.
      • Guo Y.
      • Liu F.
      • Yao Y.
      • Li P.
      • Wang D.
      • Wang Z.
      • Chen Y.
      • Ran H.
      Drug release from phase-changeable nanodroplets triggered by low-intensity focused ultrasound.
      ) and nanocups (
      • Myers R.
      • Coviello C.
      • Erbs P.
      • Foloppe J.
      • Rowe C.
      • Kwan J.
      • Crake C.
      • Finn S.
      • Jackson E.
      • Balloul J.M.
      • Story C.
      • Coussios C.
      • Carlisle R.
      Polymeric cups for cavitation-mediated delivery of oncolytic vaccinia virus.
      ); for BBB opening (nanodroplets;
      • Wu S.Y.
      • Fix S.M.
      • Arena C.B.
      • Chen C.C.
      • Zheng W.
      • Olumolade O.O.
      • Papadopoulou V.
      • Novell A.
      • Dayton P.A.
      • Konofagou E.E.
      Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: Vaporization efficiency dictates large molecular delivery.
      ); and for sonobactericide (nanodroplets;
      • Guo H.
      • Wang Z.
      • Du Q.
      • Li P.
      • Wang Z.
      • Wang A.
      Stimulated phase-shift acoustic nanodroplets enhance vancomycin efficacy against methicillin-resistant Staphylococcus aureus biofilms.
      ).

      Bubble–cell interaction

      Physics

      The physics of the interaction between bubbles or droplets and cells are described as these are the main cavitation nuclei used for drug delivery and therapy.

      Physics of microbubble–cell interaction

      Being filled with gas and/or vapor makes bubbles highly responsive to changes in pressure, and hence, exposure to ultrasound can cause rapid and dramatic changes in their volume. These volume changes in turn give rise to an array of mechanical, thermal and chemical phenomena that can significantly influence the bubbles’ immediate environment and mediate therapeutic effects. For the sake of simplicity, these phenomena are discussed in the context of a single bubble. It is important to note, however, that biological effects are typically produced by a population of bubbles and the influence of inter-bubble interactions should not be neglected.

      Mechanical effects

      A bubble in a liquid is subject to multiple competing influences: the driving pressure of the imposed ultrasound field; the hydrostatic pressure imposed by the surrounding liquid; the pressure of the gas and/or vapor inside the bubble; surface tension and the influence of any coating material; the inertia of the surrounding fluid; and damping caused by the viscosity of the surrounding fluid and/or coating, thermal conduction and/or acoustic radiation.
      The motion of the bubble is determined primarily by the competition between the liquid inertia and the internal gas pressure. This competition can be characterized by using the Rayleigh–Plesset equation for bubble dynamics to compare the relative contributions of the terms describing inertia and pressure to the acceleration of the bubble wall (
      • Flynn H.G.
      Cavitation dynamics: I. Mathematical formulation.
      ):
      R¨=(32R˙2R)+(pG(R)+p(t)2σRρLR)=IF+PF
      (1)


      where R is the time-dependent bubble radius with initial value Ro, pG is the pressure of the gas inside the bubble, p is the combined hydrostatic and time-varying pressure in the liquid, σ is the surface tension at the gas–liquid interface, ρL is the liquid density, IF is inertia factor and PF the pressure factor.
      • Flynn H.G.
      Cavitation dynamics: I. Mathematical formulation.
      ,
      • Flynn H.G.
      Cavitation dynamics: II. Free pulsations and models for cavitation bubbles.
      ) identified two scenarios: If the PF is dominant when the bubble approaches its minimum size, then the bubble will undergo sustained volume oscillations. If the inertia term is dominant (IF), then the bubble will undergo inertial collapse, similar to an empty cavity, after which it may rebound or it may disintegrate. Which of these scenarios occurs is dependent upon the bubble expansion ratio Rmax/Ro and, hence, the bubble size and the amplitude and frequency of the applied ultrasound field.
      Both inertial and non-inertial bubble oscillations can give rise to multiple phenomena that affect the bubble's immediate environment and hence are important for therapy. These include:
      • 1.
        Direct impingement: Even at moderate amplitudes of oscillation, the acceleration of the bubble wall may be sufficient to impose significant forces on nearby surfaces, easily deforming fragile structures such as biological cell membranes (
        • van Wamel A.
        • Kooiman K.
        • Harteveld M.
        • Emmer M.
        • ten Cate F.J.
        • Versluis M.
        • de Jong N.
        Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation.
        ;
        • Kudo N.
        High-Speed In Situ Observation System for Sonoporation of Cells With Size- and Position-Controlled Microbubbles.
        ) and blood vessel walls (
        • Chen H.
        • Brayman A.A.
        • Kreider W.
        • Bailey M.R.
        • Matula T.J.
        Observations of translation and jetting of ultrasound-activated microbubbles in mesenteric microvessels.
        ).
      • 2.
        Ballistic motion: In addition to oscillating, the bubble may undergo translation as a result of the pressure gradient in the fluid generated by a propagating ultrasound wave (primary radiation force). Because of their high compressibility, bubbles may travel at significant velocities, sufficient to push them toward targets for improved local deposition of a drug (
        • Dayton P.
        • Klibanov A.
        • Brandenburger G.
        • Ferrara K.
        Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles.
        ) or to penetrate biological tissue (
        • Caskey C.F.
        • Qin S.
        • Dayton P.A.
        • Ferrara K.W.
        Microbubble tunneling in gel phantoms.
        ;
        • Bader K.B.
        • Gruber M.J.
        • Holland C.K.
        Shaken and stirred: Mechanisms of ultrasound-enhanced thrombolysis.
        ;
        • Acconcia C.N.
        • Leung B.Y.
        • Goertz D.E.
        The microscale evolution of the erosion front of blood clots exposed to ultrasound stimulated microbubbles.
        ).
      • 3.
        Microstreaming: When a structure oscillates in a viscous fluid there will be a transfer of momentum as a result of interfacial friction. Any asymmetry in the oscillation will result in a net motion of that fluid in the immediate vicinity of the structure known as microstreaming (
        • Kolb J.
        • Nyborg W.L.
        Small-scale acoustic streaming in liquids.
        ). This motion will in turn impose shear stresses upon any nearby surfaces, as well as increase convection within the fluid. Because of the inherently non-linear nature of bubble oscillations (eqn [1]), both non-inertial and inertial cavitation can produce significant microstreaming, resulting in fluid velocities on the order of 1 mm/s (

        Pereno V, Characterisation of microbubble-membrane interactions in ultrasound mediated drug delivery. D.Phil. Thesis, University of Oxford; 2018. https://ora.ox.ac.uk/objects/uuid:515f2c15-e9d3-46b8-875c-420084fbc9a3

        ). If the bubble is close to a surface then it will also exhibit non-spherical oscillations, which increases the asymmetry and hence the microstreaming even further (
        • Nyborg W.L.
        Acoustic streaming near a boundary.
        ;
        • Marmottant P.
        • Hilgenfeldt S.
        Controlled vesicle deformation and lysis by single oscillating bubbles.
        ).
      • 4.
        Microjetting: Another phenomenon associated with non-spherical bubble oscillations near a surface is the generation of a liquid jet during bubble collapse. If there is sufficient asymmetry in the acceleration of the fluid on either side of the collapsing bubble, then the more rapidly moving fluid may deform the bubble into a toroidal shape, causing a high-velocity jet to be emitted on the opposite side. Microjetting has been reported to be capable of producing pitting even in highly resilient materials such as steel (
        • Naudé C.F.
        • Ellis A.T.
        On the mechanism of cavitation damage by nonhemispherical cavities collapsing in contact with a solid boundary.
        ;
        • Benjamin T.B.
        • Ellis A.T.
        The collapse of cavitation bubbles and the pressures thereby produced against solid boundaries.
        ). However, as both the direction and velocity of the jet are determined by the elastic properties of the nearby surface, its effects in biological tissue are more difficult to predict (
        • Kudo N.
        • Kinoshita Y.
        Effects of cell culture scaffold stiffness on cell membrane damage induced by sonoporation.
        ). Nevertheless, as reported by
        • Chen H.
        • Brayman A.A.
        • Kreider W.
        • Bailey M.R.
        • Matula T.J.
        Observations of translation and jetting of ultrasound-activated microbubbles in mesenteric microvessels.
        , in many cases a bubble will be sufficiently confined that microjetting will have an impact on surrounding structures regardless of jet direction.
      • 5.
        Shock waves: An inertially collapsing cavity that results in supersonic bubble wall velocities creates a significant discontinuity in the pressure in the surrounding liquid leading to the emission of a shock wave, which may impose significant stresses on nearby structures.
      • 6.
        Secondary radiation force: At smaller amplitudes of oscillation, a bubble will also generate a pressure wave in the surrounding fluid. If the bubble is adjacent to a surface, interaction between this wave and its reflection from the surface leads to a pressure gradient in the liquid and a secondary radiation force on the bubble. As with microjetting, the elastic properties of the boundary will determine the phase difference between the radiated and reflected waves and, hence, whether the bubbles move toward or away from the surface. Motion toward the surface may amplify the effects of phenomena 1, 3 and 6.

      Thermal effects

      As described above, an oscillating microbubble will re-radiate energy from the incident ultrasound field in the form of a spherical pressure wave. In addition, the non-linear character of the microbubble oscillations will lead to the re-radiation of energy over a range of frequencies. At moderate driving pressures, the bubble spectrum will contain integer multiples (harmonics) of the driving frequency; and at higher pressures, also fractional components (sub- and ultraharmonics). In biological tissue, absorption of ultrasound increases with frequency and this non-linear behavior thus also increases the rate of heating (
      • Hilgenfeldt S.
      • Lohse D.
      • Zomack M.
      Sound scattering and localized heat deposition of pulse-driven microbubbles.
      ;
      • Holt R.G.
      • Roy R.A.
      Measurements of bubble-enhanced heating from focused, MHz-frequency ultrasound in a tissue-mimicking material.
      ). Bubbles will also dissipate energy as a result of viscous friction in the liquid and thermal conduction from the gas core, the temperature of which increases during compression. Which mechanism is dominant depends on the size of the bubble, the driving conditions and the viscosity of the medium. Thermal damping is, however, typically negligible in biomedical applications of ultrasound as the time constant associated with heat transfer is much longer than the period of the microbubble oscillations (
      • Prosperetti A.
      Thermal effects and damping mechanisms in forced radial oscillations of gas-bubbles in liquids.
      ).

      Chemical effects

      The temperature rise produced in the surrounding tissue will be negligible compared with that occurring inside the bubble, especially during inertial collapse when it may reach several thousand Kelvin (
      • Flint E.B.
      • Suslick K.S.
      The temperature of cavitation.
      ). The gas pressure similarly increases significantly. Although only sustained for a very brief period, these extreme conditions can produce highly reactive chemical species, in particular reactive oxygen species (ROS), as well as the emission of electromagnetic radiation (sonoluminescence). ROS have been reported to play a significant role in multiple biological processes (
      • Winterbourn C.C.
      Reconciling the chemistry and biology of reactive oxygen species.
      ), and both ROS and sonoluminescence may affect drug activity (
      • Rosenthal I.
      • Sostaric J.Z.
      • Riesz P.
      Sonodynamic therapy—A review of the synergistic effects of drugs and ultrasound.
      ;
      • Trachootham D.
      • Alexandre J.
      • Huang P.
      Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?.
      ;
      • Beguin E.
      • Shrivastava S.
      • Dezhkunov N.V.
      • McHale A.P.
      • Callan J.F.
      • Stride E.
      Direct evidence of multibubble sonoluminescence using therapeutic ultrasound and microbubbles.
      ).

      Physics of droplet–cell interaction

      Droplets consist of an encapsulated quantity of a volatile liquid, such as perfluorobutane (boiling point: –1.7°C) or perfluoropentane (boiling point: 29°C), which is in a superheated state at body temperature. Superheated state means that although the volatile liquids have a boiling point below 37°C, these droplets remain in the liquid phase and do not exhibit spontaneous vaporization after injection. Vaporization can be achieved instead by exposure to ultrasound of significant amplitude via a process known as acoustic droplet vaporization (ADV) (
      • Kripfgans O.D.
      • Fowlkes J.B.
      • Miller D.L.
      • Eldevik O.P.
      • Carson P.L.
      Acoustic droplet vaporization for therapeutic and diagnostic applications.
      ). Before vaporization, the droplets are typically one order of magnitude smaller than the emerging bubbles, and the perfluorocarbon is inert and biocompatible (
      • Biro G.P.
      • Blais P.
      Perfluorocarbon blood substitutes.
      ). These properties enable a range of therapeutic possibilities (
      • Sheeran P.S.
      • Dayton P.A.
      Phase-change contrast agents for imaging and therapy.
      ;
      • Lea-Banks H.
      • O'Reilly M.A.
      • Hynynen K.
      Ultrasound-responsive droplets for therapy: A review.
      ). For example, unlike microbubbles, small droplets may extravasate from the leaky vessels into tumor tissue because of the enhanced permeability and retention (EPR) effect (
      • Long D.M.
      • Multer F.K.
      • Greenburg A.G.
      • Peskin G.W.
      • Lasser E.C.
      • Wickham W.G.
      • Sharts C.M.
      Tumor imaging with x-rays using macrophage uptake of radiopaque fluorocarbon emulsions.
      ;
      • Lammers T.
      • Kiessling F.
      • Hennink W.E.
      • Storm G.
      Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress.
      ;
      • Maeda H.
      Macromolecular therapeutics in cancer treatment: The EPR effect and beyond.
      ), and then be turned into bubbles by ADV (
      • Rapoport N.Y.
      • Kennedy A.M.
      • Shea J.E.
      • Scaife C.L.
      • Nam K.H.
      Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles.
      ;
      • Kopechek J.A.
      • Park E.
      • Mei C.S.
      • McDannold N.J.
      • Porter T.M.
      Accumulation of phase-shift nanoemulsions to enhance MR-guided ultrasound-mediated tumor ablation in vivo.
      ). Loading the droplets with a drug enables local delivery (
      • Rapoport N.Y.
      • Kennedy A.M.
      • Shea J.E.
      • Scaife C.L.
      • Nam K.H.
      Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles.
      ) by way of ADV. The mechanism behind this is that the emerging bubbles give rise to similar radiation forces and microstreaming as described earlier in the Physics of the Microbubble–Cell Interaction. It should be noted that oxygen is taken up during bubble growth (
      • Radhakrishnan K.
      • Holland C.K.
      • Haworth K.J.
      Scavenging dissolved oxygen via acoustic droplet vaporization.
      ), which could lead to hypoxia.
      The physics of the droplet–cell interaction is largely governed by the ADV. In general, it has been observed that ADV is promoted by the following factors: large peak negative pressures (
      • Kripfgans O.D.
      • Fowlkes J.B.
      • Miller D.L.
      • Eldevik O.P.
      • Carson P.L.
      Acoustic droplet vaporization for therapeutic and diagnostic applications.
      ), usually obtained by strong focusing of the generated beam, high frequency of the emitted wave and a relatively long distance between the transducer and the droplet. Another observation that has been made with micrometer-sized droplets is that vaporization often starts at a well-defined nucleation spot near the side of the droplet where the acoustic wave impinges (
      • Shpak O.
      • Verweij M.
      • Vos H.J.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Acoustic droplet vaporization is initiated by superharmonic focusing.
      ). These facts can be explained by considering the two mechanisms that play a role in achieving a large peak negative pressure inside the droplet: acoustic focusing and non-linear ultrasound propagation (
      • Shpak O.
      • Verweij M.
      • de Jong N.
      • Versluis M.
      Droplets, bubbles and ultrasound interactions.
      ). In the following, lengths and sizes are related to the wavelength, that is, the distance traveled by a wave in one oscillation (e.g., a 1-MHz ultrasound wave that is traveling in water with a wave speed, c, of 1500 m/s has a wavelength, w (m), of c/f = 1500/106 = 0.0015, that is, 1.5 mm.

      Acoustic focusing

      Because the speed of sound in perfluorocarbon liquids is significantly lower than that in water or tissue, refraction of the incident wave will occur at the interface between these fluids, and the spherical shape of the droplet will give rise to focusing. The assessment of this focusing effect is not straightforward because the traditional way of describing these phenomena with rays that propagate along straight lines (the ray approach) holds only for objects that are much larger than the applied wavelength. In the current case, the frequency of a typical ultrasound wave used for insonification is in the order of 1–5 MHz, yielding wavelengths in the order of 1500–300 µm, while a droplet will be smaller by two to four orders of magnitude. In addition, using the ray approach, the lower speed of sound in perfluorocarbon would yield a focal spot near the backside of the droplet, which is in contradiction to observations. The correct way to treat the focusing effect is to solve the full diffraction problem by decomposing the incident wave, the wave reflected by the droplet and the wave transmitted into the droplet into a series of spherical waves. For each spherical wave, the spherical reflection and transmission coefficients can be derived. Superposition of all the spherical waves yields the pressure inside the droplet. Nevertheless, when this approach is only applied to an incident wave with the frequency that is emitted by the transducer, this will lead neither to the right nucleation spot nor to sufficient negative pressure for vaporization. Nanoscale droplets may be too small to make effective use of the focusing mechanism, and ADV is therefore less dependent on the frequency.

      Non-linear ultrasound propagation

      High pressure amplitudes, high frequencies and long propagation distances all promote non-linear propagation of an acoustic wave (
      • Hamilton M.F.
      • Blackstock D.T.
      Nonlinear acoustics.
      ). In the time domain, non-linear propagation manifests as an increasing deformation of the shape of the ultrasound wave with distance traveled. In the frequency domain, this translates to increasing harmonic content, that is, frequencies that are multiples of the driving frequency. The total incident acoustic pressure p(t) at the position of a nanodroplet can therefore be written as
      p(t)=n=1ancos(nωt+ϕn)
      (2)


      where n is the number of a harmonic, an and ϕn are the amplitude and phase of this harmonic and ω is the angular frequency of the emitted wave. The wavelength of a harmonic wave is a fraction of the emitted wavelength.
      The aforementioned effects are both important in the case of ADV and should therefore be combined. This implies that first the amplitudes and phases of the incident non-linear ultrasound wave at the droplet location should be computed. Next, for each harmonic, the diffraction problem should be solved in terms of spherical harmonics. Adding the diffracted waves inside the droplet with the proper amplitude and phase will then yield the total pressure in the droplet. Figure 1 illustrates that the combined effects of non-linear propagation and diffraction can cause a dramatic amplification of the peak negative pressure in the micrometer-sized droplet, sufficient for triggering droplet vaporization (
      • Shpak O.
      • Verweij M.
      • Vos H.J.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Acoustic droplet vaporization is initiated by superharmonic focusing.
      ). Moreover, the location of the negative pressure peak also agrees with the observed nucleation spot.
      Fig 1
      Fig. 1Combined effect of non-linear propagation and focusing of the harmonics in a perfluoropentane micrometer-sized droplet. The emitted ultrasound wave has a frequency of 3.5 MHz and a focus at 3.81 cm, and the radius of the droplet is 10 µm for ease of observation. The pressures are given on the axis of the droplet along the propagating direction of the ultrasound wave, and the shaded area indicates the location of the droplet. Reprinted with permission from
      • Shpak O.
      • Verweij M.
      • Vos H.J.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Acoustic droplet vaporization is initiated by superharmonic focusing.
      .
      After vaporization has started, the growth of the emerging bubble is limited by inertia and heat transfer. In the absence of the heat transfer limitation, the inertia of the fluid that surrounds the bubble limits the rate of bubble growth, which is linearly proportional to time and inversely proportional to the square root of the density of the surrounding fluid. When inertia is neglected, thermal diffusion is the limiting factor in the transport of heat to drive the endothermic vaporization process of perfluorocarbon, causing the radius of the bubble to increase with the square root of time. In reality, both processes occur simultaneously, where the inertia effect is dominant at the early stage and the diffusion effect is dominant at the later stage of bubble growth. The final size that is reached by a bubble depends on the time that a bubble can expand, that is, on the duration of the negative cycle of the insonifying pressure wave. It is therefore expected that lower insonification frequencies give rise to larger maximum bubble size. Thus, irrespective of their influence on triggering ADV, lower frequencies would lead to more violent inertial cavitation effects and cause more biological damage, as experimentally observed for droplets with a radius in the order of 100 nm (
      • Burgess M.T.
      • Porter T.M.
      Control of acoustic cavitation for efficient sonoporation with phase-shift nanoemulsions.
      ).

      Biological mechanisms and bio-effects of ultrasound-activated cavitation nuclei

      The biological phenomena of sonoporation (i.e., membrane pore formation), stimulated endocytosis and opening of cell–cell contacts and the bio-effects of intracellular calcium transients, ROS generation, cell membrane potential change and cytoskeleton changes have been observed for several years (
      • Sutton J.T.
      • Haworth K.J.
      • Pyne-Geithman G.
      • Holland C.K.
      Ultrasound-mediated drug delivery for cardiovascular disease.
      ;
      • Kooiman K.
      • Vos H.J.
      • Versluis M.
      • de Jong N.
      Acoustic behavior of microbubbles and implications for drug delivery.
      ;
      • Lentacker I.
      • De Cock I.
      • Deckers R.
      • De Smedt S.C.
      • Moonen C.T.
      Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms.
      ;
      • Qin P.
      • Han T.
      • Yu A.C.H.
      • Xu L.
      Mechanistic understanding the bioeffects of ultrasound-driven microbubbles to enhance macromolecule delivery.
      ). However, other bio-effects induced by ultrasound-activated cavitation nuclei have recently been discovered. These include membrane blebbing as a recovery mechanism for reversible sonoporation (both for ultrasound-activated microbubbles [
      • Leow R.S.
      • Wan J.M.
      • Yu A.C.
      Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles.
      ] and upon ADV [
      • Qin D.
      • Zhang L.
      • Chang N.
      • Ni P.
      • Zong Y.
      • Bouakaz A.
      • Wan M.
      • Feng Y.
      In situ observation of single cell response to acoustic droplet vaporization: Membrane deformation, permeabilization, and blebbing.
      ]), extracellular vesicle formation (
      • Yuana Y.
      • Jiang L.
      • Lammertink B.H.A.
      • Vader P.
      • Deckers R.
      • Bos C.
      • Schiffelers R.M.
      • Moonen C.T.
      Microbubbles-assisted ultrasound triggers the release of extracellular vesicles.
      ), suppression of efflux transporter P-glycoprotein (
      • Cho H.
      • Lee H.Y.
      • Han M.
      • Choi J.R.
      • Ahn S.
      • Lee T.
      • Chang Y.
      • Park J.
      Localized down-regulation of P-glycoprotein by focused ultrasound and microbubbles induced blood-brain barrier disruption in rat brain.
      ;
      • Aryal M.
      • Fischer K.
      • Gentile C.
      • Gitto S.
      • Zhang Y.Z.
      • McDannold N.
      Effects on P-glycoprotein expression after blood-brain barrier disruption using focused ultrasound and microbubbles.
      ) and BBB (blood–brain barrier) transporter genes (
      • McMahon D.
      • Mah E.
      • Hynynen K.
      Angiogenic response of rat hippocampal vasculature to focused ultrasound-mediated increases in blood-brain barrier permeability.
      ). At the same time, more insight has been gained into the origin of the bio-effects, largely through the use of live cell microscopy. For sonoporation, real-time membrane pore opening and closure dynamics were revealed with pores <30 µm2 closing within 1 min, while pores >100 µm2 did not reseal (
      • Hu Y.
      • Wan J.M.
      • Yu A.C.
      Membrane perforation and recovery dynamics in microbubble-mediated sonoporation.
      ) as well as immediate rupture of filamentary actin at the pore location (
      • Chen X.
      • Leow R.S.
      • Hu Y.
      • Wan J.M.
      • Yu A.C.
      Single-site sonoporation disrupts actin cytoskeleton organization.
      ) and correlation of intracellular ROS levels with the degree of sonoporation (
      • Jia C.
      • Xu L.
      • Han T.
      • Cai P.
      • Yu A.C.H.
      • Qin P.
      Generation of reactive oxygen species in heterogeneously sonoporated cells by microbubbles with single-pulse ultrasound.
      ). Real-time sonoporation and opening of cell–cell contacts in the same endothelial cells have been reported as well for a single example (
      • Helfield B.
      • Chen X.
      • Watkins S.C.
      • Villanueva F.S.
      Biophysical insight into mechanisms of sonoporation.
      ). The applied acoustic pressure was found to determine uptake of model drugs via sonoporation or endocytosis in another study (
      • De Cock I.
      • Zagato E.
      • Braeckmans K.
      • Luan Y.
      • de Jong N.
      • De Smedt S.C.
      • Lentacker I.
      Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis.
      ). Electron microscopy revealed formation of transient membrane disruptions and permanent membrane structures, that is, caveolar endocytic vesicles, upon ultrasound and microbubble treatment (
      • Zeghimi A.
      • Escoffre J.M.
      • Bouakaz A.
      Role of endocytosis in sonoporation-mediated membrane permeabilization and uptake of small molecules: An electron microscopy study.
      ). A study by
      • Fekri F.
      • Delos Santos R.C.
      • Karshafian R.
      • Antonescu C.N.
      Ultrasound microbubble treatment enhances clathrin-mediated endocytosis and fluid-phase uptake through distinct mechanisms.
      revealed that enhanced clathrin-mediated endocytosis and fluid-phase endocytosis occur through distinct signaling mechanisms upon ultrasound and microbubble treatment. The majority of these bio-effects have been observed in in vitro models using largely non-endothelial cells and may therefore not be directly relevant to in vivo tissue, where intravascular micron-sized cavitation nuclei will only have contact with endothelial cells and circulating blood cells. On the other hand, the mechanistic studies by
      • Belcik J.T.
      • Mott B.H.
      • Xie A.
      • Zhao Y.
      • Kim S.
      • Lindner N.J.
      • Ammi A.
      • Linden J.M.
      • Lindner J.R.
      Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.
      ,
      • Belcik J.T.
      • Davidson B.P.
      • Xie A.
      • Wu M.D.
      • Yadava M.
      • Qi Y.
      • Liang S.
      • Chon C.R.
      • Ammi A.Y.
      • Field J.
      • Harmann L.
      • Chilian W.M.
      • Linden J.
      • Lindner J.R.
      Augmentation of muscle blood flow by ultrasound cavitation is mediated by ATP and purinergic signaling.
      ) and
      • Yu F.T.H.
      • Chen X.
      • Straub A.C.
      • Pacella J.J.
      The role of nitric oxide during sonoreperfusion of microvascular obstruction.
      do reveal translation from in vitro to in vivo. In these studies, ultrasound-activated microbubbles were found to induce a shear-dependent increase in intravascular adenosine triphosphate (ATP) from both endothelial cells and erythrocytes, an increase in intramuscular nitric oxide and downstream signaling through both nitric oxide and prostaglandins, which resulted in augmentation of muscle blood flow. Ultrasound settings were similar, namely, 1.3 MHz, mechanical index (MI) 1.3 for
      • Belcik J.T.
      • Mott B.H.
      • Xie A.
      • Zhao Y.
      • Kim S.
      • Lindner N.J.
      • Ammi A.
      • Linden J.M.
      • Lindner J.R.
      Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.
      ,
      • Belcik J.T.
      • Davidson B.P.
      • Xie A.
      • Wu M.D.
      • Yadava M.
      • Qi Y.
      • Liang S.
      • Chon C.R.
      • Ammi A.Y.
      • Field J.
      • Harmann L.
      • Chilian W.M.
      • Linden J.
      • Lindner J.R.
      Augmentation of muscle blood flow by ultrasound cavitation is mediated by ATP and purinergic signaling.
      ) and 1 MHz, MI 1.5 for
      • Yu F.T.H.
      • Chen X.
      • Straub A.C.
      • Pacella J.J.
      The role of nitric oxide during sonoreperfusion of microvascular obstruction.
      , with MI defined as MI = P/ f, where P_ is the derated peak negative pressure of the ultrasound wave (in MPa) and f the center frequency of the ultrasound wave (in MHz).
      Whether or not there is a direct relationship between the type of microbubble oscillation and specific bio-effects remains to be elucidated, although more insight has been gained through ultrahigh-speed imaging of the microbubble behavior in conjunction with live cell microscopy. For example, there seems to be a microbubble excursion threshold above which sonoporation occurs (
      • Helfield B.
      • Chen X.
      • Watkins S.C.
      • Villanueva F.S.
      Biophysical insight into mechanisms of sonoporation.
      ).
      • van Rooij T.
      • Skachkov I.
      • Beekers I.
      • Lattwein K.R.
      • Voorneveld J.D.
      • Kokhuis T.J.
      • Bera D.
      • Luan Y.
      • van der Steen A.F.
      • de Jong N.
      • Kooiman K.
      Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.
      further found that displacement of targeted microbubbles enhanced reversible sonoporation and preserved cell viability, whilst microbubbles that did not displace were identified as the main contributors to cell death.
      All of the aforementioned biological observations, mechanisms and effects relate to eukaryotic cells. Study of the biological effects of cavitation on, for example, bacteria is in its infancy, but studies suggest that sonoporation can be achieved in Gram-negative bacteria, with dextran uptake and gene transfection being reported in Fusobacterium nucleatum (
      • Han Y.W.
      • Ikegami A.
      • Chung P.
      • Zhang L.
      • Deng C.X.
      Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum.
      ). More recent studies have investigated the effect of microbubbles and ultrasound on gene expression (
      • Li S.
      • Zhu C.
      • Fang S.
      • Zhang W.
      • He N.
      • Xu W.
      • Kong R.
      • Shang X.
      Ultrasound microbubbles enhance human beta-defensin 3 against biofilms.
      ;
      • Dong Y.
      • Xu Y.
      • Li P.
      • Wang C.
      • Cao Y.
      • Yu J.
      Antibiofilm effect of ultrasound combined with microbubbles against Staphylococcus epidermidis biofilm.
      ;
      • Zhou H.
      • Fang S.
      • Kong R.
      • Zhang W.
      • Wu K.
      • Xia R.
      • Shang X.
      • Zhu C.
      Effect of low frequency ultrasound plus fluorescent composite carrier in the diagnosis and treatment of methicillin-resistant Staphylococcus aureus biofilm infection of bone joint implant.
      ). The findings are conflicting because although they all reveal a reduction in expression of genes involved in biofilm formation and resistance to antibiotics, an increase in expression of genes involved with dispersion and detachment of biofilms was also found (
      • Dong Y.
      • Xu Y.
      • Li P.
      • Wang C.
      • Cao Y.
      • Yu J.
      Antibiofilm effect of ultrasound combined with microbubbles against Staphylococcus epidermidis biofilm.
      ). This cavitation-mediated bio-effect needs further investigation.

      Modelling microbubble–cell–drug interaction

      Whilst there have been significant efforts to model the dynamics of ultrasound-driven microbubbles (
      • Faez T.
      • Emmer M.
      • Kooiman K.
      • Versluis M.
      • van der Steen A.F.
      • de Jong N.
      20 years of ultrasound contrast agent modeling.
      ;
      • Dollet B.
      • Marmottant P.
      • Garbin V.
      Bubble dynamics in soft and biological matter.
      ), less attention has been paid to the interactions between microbubbles and cells or their impact upon drug transport. Currently there are no models that describe the interactions between microbubbles, cells and drug molecules. Several models have been proposed for the microbubble–cell interaction in sonoporation focusing on different aspects: cell expansion and microbubble jet velocity (
      • Guo X.
      • Cai C.
      • Xu G.
      • Yang Y.
      • Tu J.
      • Huang P.
      • Zhang D.
      Interaction between cavitation microbubble and cell: A simulation of sonoporation using boundary element method (BEM).
      ), the shear stress exerted on the cell membrane (
      • Wu J.
      Theoretical study on shear stress generated by microstreaming surrounding contrast agents attached to living cells.
      ;
      • Doinikov A.A.
      • Bouakaz A.
      Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sonoporation.
      ;
      • Forbes M.M.
      • O'Brien Jr, W.D.
      Development of a theoretical model describing sonoporation activity of cells exposed to ultrasound in the presence of contrast agents.
      ;
      • Yu H.
      • Chen S.
      A model to calculate microstreaming-shear stress generated by oscillating microbubbles on the cell membrane in sonoporation.
      ;
      • Cowley J.
      • McGinty S.
      A mathematical model of sonoporation using a liquid-crystalline shelled microbubble.
      ), microstreaming (
      • Yu H.
      • Chen S.
      A model to calculate microstreaming-shear stress generated by oscillating microbubbles on the cell membrane in sonoporation.
      ), the shear stress exerted on the cell membrane in combination with microstreaming (
      • Li W.
      • Yuan T.
      • Xia-Sheng G.
      • Di X.
      • Dong Z.
      Microstreaming velocity field and shear stress created by an oscillating encapsulated microbubble near a cell membrane.
      ) or other flow phenomena (
      • Yu H.
      • Lin Z.
      • Xu L.
      • Liu D.
      • Shen Y.
      Theoretical study of microbubble dynamics in sonoporation.
      ;
      • Rowlatt C.F.
      • Lind S.J.
      Bubble collapse near a fluid-fluid interface using the spectral element marker particle method with applications in bioengineering.
      ) generated by an oscillating microbubble. In contrast to the other models,
      • Man V.H.
      • Truong P.M.
      • Li M.S.
      • Wang J.
      • Van-Oanh N.T.
      • Derreumaux P.
      • Nguyen P.H.
      Molecular mechanism of the cell membrane pore formation induced by bubble stable cavitation.
      propose that the microbubble-generated shear stress does not induce pore formation, but is instead due to microbubble fusion with the membrane and subsequent “pull out” of cell membrane lipid molecules by the oscillating microbubble. Models for pore formation (e.g.,
      • Koshiyama K.
      • Wada S.
      Molecular dynamics simulations of pore formation dynamics during the rupture process of a phospholipid bilayer caused by high-speed equibiaxial stretching.
      ) and resealing (
      • Zhang L.L.
      • Zhang Z.S.
      • Negahban M.
      • Jerusalem A.
      Molecular dynamics simulation of cell membrane pore sealing.
      ) in cell membranes have also been developed, but these models neglect the mechanism by which the pore is created. There is just one sonoporation dynamics model, developed by
      • Fan Z.
      • Liu H.
      • Mayer M.
      • Deng C.X.
      Spatiotemporally controlled single cell sonoporation.
      , that relates the uptake of the model drug propidium iodide (PI) to the size of the created membrane pore and the pore resealing time for a single cell in an in vitro setting. The model describes the intracellular fluorescence intensity of PI as a function of time, F(t), by
      F(t)=α·πDC0·ro·1β(1eβt)
      (3)


      where α is the coefficient that relates the amount of PI molecules to the fluorescence intensity of PI-DNA and PI-RNA, D is the diffusion coefficient of PI, C0 is the extracellular PI concentration, r0 is the initial radius of the pore, β is the pore re-sealing coefficient and t is time. The coefficient α is determined by the sensitivity of the fluorescence imaging system, and if unknown, the equation can still be used because it is the pore size coefficient, α·πDC0·r0, that determines the initial slope of the PI uptake pattern and is the scaling factor for the exponential increase. A cell with a large pore will have a steep initial slope of PI uptake, and the maximum PI intensity quickly reaches the plateau value. A limitation of this model is that eqn (3) is based on 2-D free diffusion models, which holds for PI-RNA but not for PI-DNA because the latter is confined to the nucleus. The model is independent of cell type, as Fan et al. have reported agreement with experimental results in both kidney (
      • Fan Z.
      • Liu H.
      • Mayer M.
      • Deng C.X.
      Spatiotemporally controlled single cell sonoporation.
      ) and endothelial cells (
      • Fan Z.
      • Chen D.
      • Deng C.X.
      Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.
      ). Other researchers have also used this model for endothelial cell studies and also classified the distribution of both the pore size and pore resealing coefficients using principal component analysis (PCA) to determine whether cells were reversibly or irreversibly sonoporated. In the context of BBB opening,
      • Hosseinkhah N.
      • Goertz D.E.
      • Hynynen K.
      Microbubbles and blood-brain barrier opening: A numerical study on acoustic emissions and wall stress predictions.
      have modeled the microbubble-generated shear and circumferential wall stress for 5-µm microvessels upon microbubble oscillation at a fixed MI of 0.134 for a range of frequencies (0.5, 1 and 1.5 MHz). The wall stresses were dependent upon microbubble size (range investigated: 2–18 µm in diameter) and ultrasound frequency.
      • Wiedemair W.
      • Tukovic Z.
      • Jasak H.
      • Poulikakos D.
      • Kurtcuoglu V.
      The breakup of intravascular microbubbles and its impact on the endothelium.
      have also modelled the wall shear stress generated by microbubble (2 µm in diameter) destruction at 3 MHz for larger microvessels (200 µm in diameter). The presence of red blood cells was included in the model and was found to cause confinement of pressure and shear gradients to the vicinity of the microbubble. Advances in methods for imaging microbubble–cell interactions will facilitate the development of more sophisticated mechanistic models.

      Treatment of tumors (non-brain)

      The structure of tumor tissue varies significantly from that of healthy tissue which has important implications for its treatment. To support the continuous expansion of neoplastic cells, the formation of new vessels (i.e., angiogenesis) is needed (
      • Junttila M.R.
      • de Sauvage F.J.
      Influence of tumour micro-environment heterogeneity on therapeutic response.
      ). As such, a rapidly developed, poorly organized vasculature with enlarged vascular openings arises. Between these vessels, large avascular regions exist, which are characterized by a dense extracellular matrix, high interstitial pressure, low pH and hypoxia. Moreover, a local immunosuppressive environment is formed, preventing possible anti-tumor activity by the immune system.
      Notwithstanding the growing knowledge of the pathophysiology of tumors, treatment remains challenging. Chemotherapeutic drugs are typically administered to abolish the rapidly dividing cancer cells. Yet, their cytotoxic effects are not limited to cancer cells, causing dose-limiting off-target effects. To overcome this hurdle, chemotherapeutics are often encapsulated in nano-sized carriers, that is, nanoparticles, that are designed to specifically diffuse through the large openings of tumor vasculature, while being excluded from healthy tissue by normal blood vessels (
      • Lammers T.
      • Kiessling F.
      • Hennink W.E.
      • Storm G.
      Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress.
      ;
      • Maeda H.
      Macromolecular therapeutics in cancer treatment: The EPR effect and beyond.
      ). Despite being highly promising in pre-clinical studies, drug-containing nanoparticles have exhibited limited clinical success because of the vast heterogeneity in tumor vasculature (
      • Barenholz Y.
      Doxil—The first FDA-approved nano-drug: Lessons learned.
      ;
      • Lammers T.
      • Kiessling F.
      • Hennink W.E.
      • Storm G.
      Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress.
      ;
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      ). In addition, drug penetration into the deeper layers of the tumor can be constrained by high interstitial pressure and a dense extracellular matrix in the tumor. Furthermore, acidic and hypoxic regions limit the efficacy of radiation- and chemotherapy-based treatments because of biochemical effects (
      • Mehta G.
      • Hsiao A.Y.
      • Ingram M.
      • Luker G.D.
      • Takayama S.
      Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.
      ;
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ;
      • Fix S.M.
      • Papadopoulou V.
      • Velds H.
      • Kasoji S.K.
      • Rivera J.N.
      • Borden M.A.
      • Chang S.
      • Dayton P.A.
      Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.
      ). Ultrasound-triggered microbubbles are able to alter the tumor environment locally, thereby improving drug delivery to tumors. These alterations are schematically represented in Figure 2 and include improving vascular permeability, modifying the tumor perfusion, reducing local hypoxia and overcoming the high interstitial pressure.
      Fig 2
      Fig. 2Ultrasound-activated microbubbles can locally alter the tumor microenvironment through four mechanisms: enhanced permeability, improved contact, reduced hypoxia and altered perfusion. ROS = reactive oxygen species.
      Several studies have found that ultrasound-driven microbubbles improved delivery of chemotherapeutic agents in tumors, which resulted in increased anti-tumor effects (
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      ;
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ;
      • Zhang L.
      • Yin T.H.
      • Li B.
      • Zheng R.Q.
      • Qiu C.
      • Lam K.S.
      • Zhang Q.
      • Shuai X.T.
      Size-modulable nanoprobe for high-performance ultrasound imaging and drug delivery against cancer.
      ). Moreover, several gene products could be effectively delivered to tumor cells via ultrasound-driven microbubbles, resulting in a downregulation of tumor-specific pathways and an inhibition in tumor growth (
      • Kopechek J.A.
      • Carson A.R.
      • McTiernan C.F.
      • Chen X.
      • Hasjim B.
      • Lavery L.
      • Sen M.
      • Grandis J.R.
      • Villanueva F.S.
      Ultrasound targeted microbubble destruction-mediated delivery of a transcription factor decoy inhibits STAT3 signaling and tumor growth.
      ;
      • Zhou Y.
      • Gu H.
      • Xu Y.
      • Li F.
      • Kuang S.
      • Wang Z.
      • Zhou X.
      • Ma H.
      • Li P.
      • Zheng Y.
      • Ran H.
      • Jian J.
      • Zhao Y.
      • Song W.
      • Wang Q.
      • Wang D.
      Targeted antiangiogenesis gene therapy using targeted cationic microbubbles conjugated with CD105 antibody compared with untargeted cationic and neutral microbubbles.
      ).
      • Theek B.
      • Baues M.
      • Ojha T.
      • Mockel D.
      • Veettil S.K.
      • Steitz J.
      • van Bloois L.
      • Storm G.
      • Kiessling F.
      • Lammers T.
      Sonoporation enhances liposome accumulation and penetration in tumors with low EPR.
      furthermore confirmed that nanoparticle accumulation can be achieved in tumors with low EPR effect. Drug transport and distribution through the dense tumor matrix and into regions with elevated interstitial pressure are often the limiting factors in peripheral tumors. As a result, several reports have indicated that drug penetration into the tumor remained limited after sonoporation, which may impede the eradication of the entire tumor tissue (
      • Eggen S.
      • Fagerland S.M.
      • Mørch Ý.
      • Hansen R.
      • Søvik K.
      • Berg S.
      • Furu H.
      • Bøhn A.D.
      • Lilledahl M.B.
      • Angelsen A.
      • Angelsen B.
      • de Lange Davies C.
      Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.
      ;
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      ;
      • Wei Y.L.
      • Shang N.
      • Jin H.
      • He Y.
      • Pan Y.W.
      • Xiao N.N.
      • Wei J.L.
      • Xiao S.Y.
      • Chen L.P.
      • Liu J.H.
      Penetration of different molecule sizes upon ultrasound combined with microbubbles in a superficial tumour model.
      ). Alternatively, microbubble cavitation can affect tumor perfusion, as vasoconstriction and even temporary vascular shutdown have been reported ex vivo (
      • Keravnou C.P.
      • De Cock I.
      • Lentacker I.
      • Izamis M.L.
      • Averkiou M.A.
      Microvascular injury and perfusion changes induced by ultrasound and microbubbles in a machine-perfused pig liver.
      ) and in vivo (
      • Hu X.
      • Kheirolomoom A.
      • Mahakian L.M.
      • Beegle J.R.
      • Kruse D.E.
      • Lam K.S.
      • Ferrara K.W.
      Insonation of targeted microbubbles produces regions of reduced blood flow within tumor vasculature.
      ;
      • Goertz D.E.
      An overview of the influence of therapeutic ultrasound exposures on the vasculature: High intensity ultrasound and microbubble-mediated bioeffects.
      ;
      • Yemane P.T.
      • Aslund A.
      • Saeterbo K.G.
      • Bjorkoy A.
      • Snipstad S.
      • Van Wamel A.
      • Berg S.
      • Morch Y.
      • Hansen R.
      • Angelsen B.
      • Davies C.D.
      The effect of sonication on extravasation and distribution of nanoparticles and dextrans in tumor tissue imaged by multiphoton microscopy.
      ). These effects were seen at higher ultrasound intensities (>1.5 MPa) and are believed to result from inertial cavitation leading to violent microbubble collapses. As blood supply is needed to maintain tumor growth, vascular disruption might form a different approach to cease tumor development. Microbubble-induced microvascular damage was able to complement the direct effects of chemotherapeutics and antivascular drugs by secondary ischemia-mediated cytotoxicity, which led to tumor growth inhibition (
      • Wang J.F.
      • Zhao Z.L.
      • Shen S.X.
      • Zhang C.X.
      • Guo S.C.
      • Lu Y.K.
      • Chen Y.M.
      • Liao W.J.
      • Liao Y.L.
      • Bin J.P.
      Selective depletion of tumor neovasculature by microbubble destruction with appropriate ultrasound pressure.
      ;
      • Ho Y.J.
      • Wang T.C.
      • Fan C.H.
      • Yeh C.K.
      Spatially uniform tumor treatment and drug penetration by regulating ultrasound with microbubbles.
      ;
      • Yang J.
      • Zhang X.J.
      • Cai H.J.
      • Chen Z.K.
      • Qian Q.F.
      • Xue E.S.
      • Lin L.W.
      Ultrasound-targeted microbubble destruction improved the antiangiogenic effect of Endostar in triple-negative breast carcinoma xenografts.
      ). In addition, a synergistic effect between radiation therapy and ultrasound-stimulated microbubble treatment was observed, as radiation therapy also induces secondary cell death by endothelial apoptosis and vascular damage (
      • Lai P.
      • Tarapacki C.
      • Tran W.T.
      • El Kaffas A.
      • Lee J.
      • Hupple C.
      • Iradji S.
      • Giles A.
      • Al-Mahrouki A.
      • Czarnota G.J.
      Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo.
      ;
      • Daecher A.
      • Stanczak M.
      • Liu J.B.
      • Zhang J.
      • Du S.S.
      • Forsberg F.
      • Leeper D.B.
      • Eisenbrey J.R.
      Localized microbubble cavitation-based antivascular therapy for improving HCC treatment response to radiotherapy.
      ). Nevertheless, several adverse effects have been reported because of excessive vascular disruption, including hemorrhage, tissue necrosis and the formation of thrombi (
      • Goertz D.E.
      An overview of the influence of therapeutic ultrasound exposures on the vasculature: High intensity ultrasound and microbubble-mediated bioeffects.
      ;
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      ;
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ).
      Furthermore, oxygen-containing microbubbles can provide a local oxygen supply to hypoxic areas, rendering oxygen-dependent treatments more effective. This is of interest for sonodynamic therapy, which is based on the production of cytotoxic ROS by a sonosensitizing agent upon activation by ultrasound in the presence of oxygen (
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ,
      • McEwan C.
      • Kamila S.
      • Owen J.
      • Nesbitt H.
      • Callan B.
      • Borden M.
      • Nomikou N.
      • Hamoudi R.A.
      • Taylor M.A.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Combined sonodynamic and antimetabolite therapy for the improved treatment of pancreatic cancer using oxygen loaded microbubbles as a delivery vehicle.
      ;
      • Nesbitt H.
      • Sheng Y.
      • Kamila S.
      • Logan K.
      • Thomas K.
      • Callan B.
      • Taylor M.A.
      • Love M.
      • O'Rourke D.
      • Kelly P.
      • Beguin E.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.
      ). As ultrasound can be used to stimulate the release of oxygen from oxygen-carrying microbubbles while simultaneously activating a sonosensitizer, this approach has been reported to be particularly useful for the treatment of hypoxic tumor types (
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ;
      • Nesbitt H.
      • Sheng Y.
      • Kamila S.
      • Logan K.
      • Thomas K.
      • Callan B.
      • Taylor M.A.
      • Love M.
      • O'Rourke D.
      • Kelly P.
      • Beguin E.
      • Stride E.
      • McHale A.P.
      • Callan J.F.
      Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.
      ). Additionally, low oxygenation promotes resistance to radiotherapy, which can be circumvented by a momentary supply of oxygen. Based on this notion, oxygen-carrying microbubbles were used to improve the outcome of radiotherapy in a rat fibrosarcoma model (
      • Fix S.M.
      • Papadopoulou V.
      • Velds H.
      • Kasoji S.K.
      • Rivera J.N.
      • Borden M.A.
      • Chang S.
      • Dayton P.A.
      Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.
      ).
      Finally, ultrasound-activated microbubbles promote convection and induce acoustic radiation forces. As such, closer contact with the tumor endothelium and an extended contact time can be obtained (
      • Kilroy J.P.
      • Klibanov A.L.
      • Wamhoff B.R.
      • Bowles D.K.
      • Hossack J.A.
      Localized in vivo model drug delivery with intravascular ultrasound and microbubbles.
      ). Furthermore, these forces may counteract the elevated interstitial pressure present in tumors (
      • Eggen S.
      • Fagerland S.M.
      • Mørch Ý.
      • Hansen R.
      • Søvik K.
      • Berg S.
      • Furu H.
      • Bøhn A.D.
      • Lilledahl M.B.
      • Angelsen A.
      • Angelsen B.
      • de Lange Davies C.
      Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.
      ;
      • Lea-Banks H.
      • Teo B.
      • Stride E.
      • Coussios C.C.
      The effect of particle density on ultrasound-mediated transport of nanoparticles.
      ;
      • Xiao N.
      • Liu J.
      • Liao L.
      • Sun J.
      • Jin W.
      • Shu X.
      Ultrasound combined with microbubbles increase the delivery of doxorubicin by reducing the interstitial fluid pressure.
      ).
      Apart from their ability to improve tumor uptake, microbubbles can be used as ultrasound-responsive drug carriers to reduce the off-target effects of chemotherapeutics. By loading the drugs or drug-containing nanoparticles directly into or onto the microbubbles, a spatial and temporal control of drug release can be obtained, thereby reducing exposure to other parts of the body (
      • Yan F.
      • Li L.
      • Deng Z.T.
      • Jin Q.F.
      • Chen J.J.
      • Yang W.
      • Yeh C.K.
      • Wu J.R.
      • Shandas R.
      • Liu X.
      • Zheng H.R.
      Paclitaxel–liposome–microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers.
      ;
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ). Moreover, several studies have reported improved anti-cancer effects from treatment with drug-coupled microbubbles, compared with a co-administration approach (
      • Burke C.W.
      • Alexander E.
      • Timbie K.
      • Kilbanov A.L.
      • Price R.J.
      Ultrasound-activated agents comprised of 5 FU-bearing nanoparticles bonded to microbubbles inhibit solid tumor growth and improve survival.
      ;
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ). Additionally, tumor neovasculature expresses specific surface receptors that can be targeted by specific ligands. Adding such targeting moieties to the surface of (drug-loaded) microbubbles improves site-targeted delivery and has been found to potentiate this effect further (
      • Bae Y.J.
      • Yoon Y.I.
      • Yoon T.J.
      • Lee H.J.
      Ultrasound-guided delivery of siRNA and a chemotherapeutic drug by using microbubble complexes: In vitro and in vivo evaluations in a prostate cancer model.
      ;
      • Xing L.
      • Shi Q.
      • Zheng K.
      • Shen M.
      • Ma J.
      • Li F.
      • Liu Y.
      • Lin L.
      • Tu W.
      • Duan Y.
      • Du L.
      Ultrasound-mediated microbubble destruction (UMMD) facilitates the delivery of CA19-9 targeted and paclitaxel loaded mPEG-PLGA-PLL nanoparticles in pancreatic cancer.
      ;
      • Luo W.X.
      • Wen G.
      • Yang L.
      • Tang J.
      • Wang J.G.
      • Wang J.H.
      • Zhang S.Y.
      • Zhang L.
      • Ma F.
      • Xiao L.L.
      • Wang Y.
      • Li Y.J.
      Dual-targeted and pH-sensitive doxorubicin prodrug-microbubble complex with ultrasound for tumor treatment.
      ).
      Phase-shifting droplets and gas-stabilizing solid agents (e.g., nanocups) have the unique ability to benefit from both EPR-mediated accumulation in the “leaky” parts of the tumor vasculature because of their small sizes, as well as from ultrasound-induced permeabilization of the tissue structure (
      • Zhou Y.F.
      Application of acoustic droplet vaporization in ultrasound therapy.
      ;
      • Myers R.
      • Coviello C.
      • Erbs P.
      • Foloppe J.
      • Rowe C.
      • Kwan J.
      • Crake C.
      • Finn S.
      • Jackson E.
      • Balloul J.M.
      • Story C.
      • Coussios C.
      • Carlisle R.
      Polymeric cups for cavitation-mediated delivery of oncolytic vaccinia virus.
      ;
      • Liu J.X.
      • Xu F.F.
      • Huang J.
      • Xu J.S.
      • Liu Y.
      • Yao Y.Z.
      • Ao M.
      • Li A.
      • Hao L.
      • Cao Y.
      • Hu Z.Q.
      • Ran H.T.
      • Wang Z.G.
      • Li P.
      Low-intensity focused ultrasound (LIFU)-activated nanodroplets as a theranostic agent for noninvasive cancer molecular imaging and drug delivery.
      ;
      • Zhang L.
      • Yin T.H.
      • Li B.
      • Zheng R.Q.
      • Qiu C.
      • Lam K.S.
      • Zhang Q.
      • Shuai X.T.
      Size-modulable nanoprobe for high-performance ultrasound imaging and drug delivery against cancer.
      ). Several research groups have reported tumor regression after treatment with acoustically active droplets (
      • Gupta R.
      • Shea J.
      • Scafe C.
      • Shurlygina A.
      • Rapoport N.
      Polymeric micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance.
      ;
      • van Wamel A.
      • Sontum P.C.
      • Healey A.
      • Kvale S.
      • Bush N.
      • Bamber J.
      • Davies C.D.
      Acoustic Cluster Therapy (ACT) enhances the therapeutic efficacy of paclitaxel and Abraxane for treatment of human prostate adenocarcinoma in mice.
      ;
      • Cao Y.
      • Chen Y.
      • Yu T.
      • Guo Y.
      • Liu F.
      • Yao Y.
      • Li P.
      • Wang D.
      • Wang Z.
      • Chen Y.
      • Ran H.
      Drug release from phase-changeable nanodroplets triggered by low-intensity focused ultrasound.
      ;
      • Liu J.X.
      • Xu F.F.
      • Huang J.
      • Xu J.S.
      • Liu Y.
      • Yao Y.Z.
      • Ao M.
      • Li A.
      • Hao L.
      • Cao Y.
      • Hu Z.Q.
      • Ran H.T.
      • Wang Z.G.
      • Li P.
      Low-intensity focused ultrasound (LIFU)-activated nanodroplets as a theranostic agent for noninvasive cancer molecular imaging and drug delivery.
      ) or gas-stabilizing solid particles (
      • Min H.S.
      • Son S.
      • You D.G.
      • Lee T.W.
      • Lee J.
      • Lee S.
      • Yhee J.Y.
      • Lee J.
      • Han M.H.
      • Park J.H.
      • Kim S.H.
      • Choi K.
      • Park K.
      • Kim K.
      • Kwon I.C.
      Chemical gas-generating nanoparticles for tumor-targeted ultrasound imaging and ultrasound-triggered drug delivery.
      ;
      • Myers R.
      • Coviello C.
      • Erbs P.
      • Foloppe J.
      • Rowe C.
      • Kwan J.
      • Crake C.
      • Finn S.
      • Jackson E.
      • Balloul J.M.
      • Story C.
      • Coussios C.
      • Carlisle R.
      Polymeric cups for cavitation-mediated delivery of oncolytic vaccinia virus.
      ). A different approach to the use of droplets for tumor treatment is ACT, which is based on microbubble-droplet clusters that upon ultrasound exposure, undergo a phase shift to create large bubbles that can transiently block capillaries (
      • Sontum P.
      • Kvale S.
      • Healey A.J.
      • Skurtveit R.
      • Watanabe R.
      • Matsumura M.
      • Ostensen J.
      Acoustic Cluster Therapy (ACT)—A novel concept for ultrasound mediated, targeted drug delivery.
      ). Although the mechanism behind the technique is not yet fully understood, studies have reported improved delivery and efficacy of paclitaxel and Abraxane in xenograft prostate tumor models (
      • van Wamel A.
      • Sontum P.C.
      • Healey A.
      • Kvale S.
      • Bush N.
      • Bamber J.
      • Davies C.D.
      Acoustic Cluster Therapy (ACT) enhances the therapeutic efficacy of paclitaxel and Abraxane for treatment of human prostate adenocarcinoma in mice.
      ;
      • Kotopoulis S.
      • Stigen E.
      • Popa M.
      • Safont M.M.
      • Healey A.
      • Kvåle S.
      • Sontum P.
      • Gjertsen B.T.
      • Gilja O.H.
      • McCormack E.
      Sonoporation with Acoustic Cluster Therapy (ACT) induces transient tumour volume reduction in a subcutaneous xenograft model of pancreatic ductal adenocarcinoma.
      ). Another use of droplets for tumor treatment is enhanced high-intensity focused ultrasound (HIFU)-mediated heating of tumors (
      • Kopechek J.A.
      • Park E.J.
      • Zhang Y.Z.
      • Vykhodtseva N.I.
      • McDannold N.J.
      • Porter T.M.
      Cavitation-enhanced MR-guided focused ultrasound ablation of rabbit tumors in vivo using phase shift nanoemulsions.
      ).
      Although microbubble-based drug delivery to solid tumors shows great promise, it also faces important challenges. The ultrasound parameters used in in vivo studies highly vary between research groups, and no consensus was found on the oscillation regime that is believed to be responsible for the observed effects (
      • Wang T.Y.
      • Choe J.W.
      • Pu K.
      • Devulapally R.
      • Bachawal S.
      • Machtaler S.
      • Chowdhury S.M.
      • Luong R.
      • Tian L.
      • Khuri-Yakub B.
      • Rao J.
      • Paulmurugan R.
      • Willmann J.K.
      Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer.
      ;
      • Snipstad S.
      • Berg S.
      • Morch Y.
      • Bjorkoy A.
      • Sulheim E.
      • Hansen R.
      • Grimstad I.
      • van Wamel A.
      • Maaland A.F.
      • Torp S.H.
      • de Lange Davies C.
      Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts.
      ). Moreover, longer ultrasound pulses and increased exposure times are usually applied in comparison to in vitro reports (
      • Roovers S.
      • Segers T.
      • Lajoinie G.
      • Deprez J.
      • Versluis M.
      • De Smedt S.C.
      • Lentacker I.
      The role of ultrasound-driven microbubble dynamics in drug delivery: From microbubble fundamentals to clinical translation.
      ). This could promote additional effects such as microbubble clustering and microbubble translation, which could cause local damage to the surrounding tissue as well (
      • Roovers S.
      • Lajoinie G.
      • De Cock I.
      • Brans T.
      • Dewitte H.
      • Braeckmans K.
      • Versuis M.
      • De Smedt S.C.
      • Lentacker I.
      Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.
      ). To elucidate these effects further, fundamental in vitro research remains important. Therefore, novel in vitro models that more accurately mimic the complexity of the in vivo tumor environment are currently being explored.
      • Park Y.C.
      • Zhang C.
      • Kim S.
      • Mohamedi G.
      • Beigie C.
      • Nagy J.O.
      • Holt R.G.
      • Cleveland R.O.
      • Jeon N.L.
      • Wong J.Y.
      Microvessels-on-a-chip to assess targeted ultrasound-assisted drug delivery.
      engineered a perfusable vessel-on-a-chip system and reported successful doxorubicin delivery to the endothelial cells lining this microvascular network. While such microfluidic chips could be extremely useful to study the interactions of microbubbles with the endothelial cell barrier, special care of the material of the chambers should be taken to avoid ultrasound reflections and standing waves (
      • Beekers I.
      • van Rooij T.
      • Verweij M.D.
      • Versluis M.
      • de Jong N.
      • Trietsch S.J.
      • Kooiman K.
      Acoustic characterization of a vessel-on-a-chip microfluidic system for ultrasound-mediated drug delivery.
      ). Alternatively, 3-D tumor spheroids have been used to study the effects of ultrasound and microbubble-assisted drug delivery on penetration and therapeutic effect in a multicellular tumor model (
      • Roovers S.
      • Lajoinie G.
      • Prakash J.
      • Versluis M.
      • De Smedt S.C.
      • Lentacker I.
      Liposome-loaded microbubbles and ultrasound enhance drug delivery in a 3D tumor spheroid.
      ). Apart from expanding the knowledge on microbubble–tissue interactions in detailed parametric studies in vitro, it will be crucial to obtain improved control over the microbubble behavior in vivo, and link this to the therapeutic effects. To this end, passive cavitation detection to monitor microbubble cavitation behavior in real time is currently under development, and could provide better insights in the future (
      • Choi J.J.
      • Carlisle R.C.
      • Coviello C.
      • Seymour L.
      • Coussios C.-C.
      Non-invasive and real-time passive acoustic mapping of ultrasound-mediated drug delivery.
      ;
      • Graham S.M.
      • Carlisle R.
      • Choi J.J.
      • Stevenson M.
      • Shah A.R.
      • Myers R.S.
      • Fisher K.
      • Peregrino M.B.
      • Seymour L.
      • Coussios C.C.
      Inertial cavitation to non-invasively trigger and monitor intratumoral release of drug from intravenously delivered liposomes.
      ;
      • Haworth K.J.
      • Bader K.B.
      • Rich K.T.
      • Holland C.K.
      • Mast T.D.
      Quantitative frequency-domain passive cavitation imaging.
      ). Efforts are being committed to construction of custom-built delivery systems, which can be equipped with multiple transducers allowing drug delivery guided by ultrasound imaging and/or passive cavitation detection (
      • Escoffre J.M.
      • Mannaris C.
      • Geers B.
      • Novell A.
      • Lentacker I.
      • Averkiou M.
      • Bouakaz A.
      Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery.
      ;
      • Choi J.J.
      • Carlisle R.C.
      • Coviello C.
      • Seymour L.
      • Coussios C.-C.
      Non-invasive and real-time passive acoustic mapping of ultrasound-mediated drug delivery.
      ;
      • Wang S.Y.
      • Wang C.Y.
      • Unnikrishnan S.
      • Klibanov A.L.
      • Hossack J.A.
      • Mauldin F.W.
      Optical verification of microbubble response to acoustic radiation force in large vessels with in vivo results.
      ;
      • Paris J.L.
      • Mannaris C.
      • Cabanas M.V.
      • Carlisle R.
      • Manzano M.
      • Vallet-Regi M.
      • Coussios C.C.
      Ultrasound-mediated cavitation-enhanced extravasation of mesoporous silica nanoparticles for controlled-release drug delivery.
      ).

      Clinical studies

      Pancreatic cancer

      The tolerability and therapeutic potential of improved chemotherapeutic drug delivery using microbubbles and ultrasound were first investigated for the treatment of inoperable pancreatic ductal adenocarcinoma at Haukeland University Hospital, Norway (
      • Kotopoulis S.
      • Dimcevski G.
      • Gilja O.H.
      • Hoem D.
      • Postema M.
      Treatment of human pancreatic cancer using combined ultrasound, microbubbles, and gemcitabine: A clinical case study.
      ;
      • Dimcevski G.
      • Kotopoulis S.
      • Bjanes T.
      • Hoem D.
      • Schjott J.
      • Gjertsen B.T.
      • Biermann M.
      • Molven A.
      • Sorbye H.
      • McCormack E.
      • Postema M.
      • Gilja O.H.
      A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer.
      ). In this clinical trial, gemcitabine was administered by intravenous injection over 30 min. During the last 10 min of chemotherapy, an abdominal echography was performed to locate the position of pancreatic tumor. At the end of chemotherapy, 0.5 mL of SonoVue microbubbles followed by 5 mL saline was intravenously injected every 3.5 min to ensure their presence throughout the whole sonoporation treatment. Pancreatic tumors were exposed to ultrasound (1.9 MHz, MI 0.2, 1% DC) using a 4C curvilinear probe (GE Healthcare) connected to an LOGIQ 9 clinical ultrasound scanner. The cumulative ultrasound exposure was only 18.9 s. All clinical data indicated that microbubble-mediated gemcitabine delivery did not induce any serious adverse events in comparison to chemotherapy alone. At the same time, tumor size and development were characterized according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. In addition, Eastern Cooperative Oncology Group performance status was used to monitor the therapeutic efficacy of microbubble-mediated gemcitabine delivery. All 10 patients tolerated an increased number of gemcitabine cycles compared with treatment with chemotherapy alone from historical controls (8.3 ± 6 vs. 13.8 ± 5.6 cycles, p < 0.008), thus reflecting an improved physical state. After 12 treatment cycles, one patient's tumor exhibited a twofold decrease in tumor size. This patient was excluded from this clinical trial to be treated with radiotherapy and then with pancreatectomy. In 5 of the 10 patients, the maximum tumor diameter was partially decreased from the first to last therapeutic treatment. Subsequently, a consolidative radiotherapy or a FOLFIRINOX treatment, a bolus and infusion of 5-fluorouracil, leucovorin, irinotecan and oxaliplatin, was offered to them. The median survival was significantly increased from 8.9 to 17.6 mo (p = 0.0001). Together, these results indicate that drug delivery using clinically approved microbubbles, chemotherapeutics and ultrasound is feasible and compatible with respect to clinical procedures. Nevertheless, the authors did not provide any evidence that the improved therapeutic efficacy of gemcitabine was related to an increase in intra-tumoral bioavailability of the drug. In addition, the effects of microbubble-assisted ultrasound treatment alone on tumor growth were not investigated, while recent publications describe that according to the ultrasound parameters, such treatment could induce a significant decrease in tumor volume through a reduction in tumor perfusion as described above.

      Hepatic metastases from the digestive system

      A tolerability study of chemotherapeutic delivery using microbubble-assisted ultrasound for the treatment of liver metastases from gastrointestinal tumors and pancreatic carcinoma was conducted at Beijing Cancer Hospital, China (
      • Wang Y.
      • Li Y.
      • Yan K.
      • Shen L.
      • Yang W.
      • Gong J.
      • Ding K.
      Clinical study of ultrasound and microbubbles for enhancing chemotherapeutic sensitivity of malignant tumors in digestive system.
      ). Thirty minutes after intravenous infusion of chemotherapy (for both monotherapy and combination therapy), 1 mL of SonoVue microbubbles was intravenously administered and was repeated another five times in 20 min. An ultrasound probe (C1-5 abdominal convex probe; GE Healthcare, USA) was positioned on the tumor lesion, which was exposed to ultrasound at different MIs (0.4–1) in contrast mode using a LogiQ E9 scanner (GE Healthcare, USA). The primary aims of this clinical trial were to evaluate the tolerability of this therapeutic procedure and to explore the largest MI and ultrasound treatment time that cancer patients can tolerate. According to the clinical tolerability evaluation, all 12 patients exhibited no serious adverse events. The authors reported that the microbubble-mediated chemotherapy led to fever in 2 patients. However, there is no clear evidence this is related to the microbubble and ultrasound treatment. Indeed, in the absence of direct comparison of these results with a historical group of patients receiving the chemotherapy on its own, one cannot rule out a direct link between the fever and the chemotherapy alone. All adverse side effects were resolved with symptomatic medication. In addition, the severity of side effects did not worsen with increases in MI, suggesting that microbubble-mediated chemotherapy is a tolerable procedure. The secondary aims were to assess the efficacy of this therapeutic protocol using contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI). Thus, tumor size and development were characterized according to the RECIST criteria. Half of the patients had stable disease, and one patient obtained a partial response after the first treatment cycle. The median progression-free survival was 91 d. However, comparison and interpretation of results are very difficult because none of the patients were treated with the same chemotherapeutics, MI and/or number of treatment cycles. The results of tolerability and efficacy evaluations should be compared with those for patients receiving the chemotherapy on its own to clearly identify the therapeutic benefit of combining therapy with ultrasound-driven microbubbles. Similar to the pancreatic clinical study, no direct evidence of enhanced therapeutic bioavailability of the chemotherapeutic drug after the treatment was provided. This investigation is all the more important as the ultrasound and microbubble treatment was applied 30 min after intravenous chemotherapy (for both monotherapy and combination therapy) independently of drug pharmacokinetics and metabolism.

      Ongoing and upcoming clinical trials

      Currently, two clinical trials are ongoing: (i) Professor F. Kiessling (RWTH Aachen University, Germany) proposes examining whether the exposure of early primary breast cancer to microbubble-assisted ultrasound during neoadjuvant chemotherapy results in increased tumor regression in comparison to that after ultrasound treatment alone (NCT03385200). (ii) Dr. J. Eisenbrey (Sidney Kimmel Cancer Center, Thomas Jefferson University, USA) is investigating the therapeutic potential of perflutren protein type A microspheres in combination with microbubble-assisted ultrasound in radioembolization therapy of liver cancer (NCT03199274).
      A proof of concept study (NCT03458975) has been set in Tours Hospital, France, for treating non-resectable liver metastases. The aim of this trial is to perform a feasibility study with the development of a dedicated ultrasound imaging and delivery probe with a therapy protocol optimized for patients with hepatic metastases of colorectal cancer and who are eligible for monoclonal antibodies in combination with chemotherapy. A dedicated 1.5-D ultrasound probe has been developed and interconnected to a modified Aixplorer imaging platform (Supersonic Imagine, Aix-en-Provence, France). The primary objective of the study is to determine the rate of objective response at 2 mo for lesions receiving optimized and targeted delivery of systemic chemotherapy combining bevacizumab and FOLFIRI compared with those treated with only the systemic chemotherapy regimen. The secondary objective is to determine the tolerability of this local approach of optimized intra-tumoral drug delivery during the 3 mo of follow-up, by assessing tumor necrosis, tumor vascularity and pharmacokinetics of bevacizumab and by profiling cytokine expression spatially.

      Immunotherapy

      Cancer immunotherapy is considered to be one of the most promising strategies to eradicate cancer as it makes use of the patient's own immune system to selectively attack and destroy tumor cells. It is a common name that refers to a variety of strategies that aim to unleash the power of the immune system by either boosting antitumoral immune responses or flagging tumor cells to make them more visible to the immune system. The principle is that tumors express specific tumor antigens which are not expressed or expressed to a much lesser extent by normal somatic cells and hence can be used to initiate a cancer-specific immune response. In this section we aim to give insight into how microbubbles and ultrasound have been applied as useful tools to initiate or sustain different types of cancer immunotherapy, as illustrated in Figure 3.
      Fig 3
      Fig. 3Schematic overview of how microbubbles (MB) and ultrasound (US) have been found to contribute to cancer immunotherapy. From left to right: Microbubbles can be used as antigen carriers to stimulate antigen uptake by dendritic cells. Microbubbles and ultrasound can alter the permeability of tumors, thereby increasing the intra-tumoral penetration of adoptively transferred immune cells or checkpoint inhibitors. Finally, exposing tissues to cavitating microbubbles can induce sterile inflammation by the local release of damage-associated molecular patterns (DAMPS).
      When Ralph Steinman (
      • Steinman R.M.
      • Kaplan G.
      • Witmer M.D.
      • Cohn Z.A.
      Identification of a novel cell type in peripheral lymphoid organs of mice: V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.
      ) discovered the dendritic cell (DC) in 1973, its central role in the initiation of immunity made it an attractive target to evoke specific antitumoral immune responses. Indeed, these cells very efficiently capture antigens and present them to T lymphocytes in major histocompatibility complexes (MHCs), thereby bridging the innate and adaptive immune systems. More specifically, exogenous antigens engulfed via the endolysosomal pathway are largely presented to CD4+ T cells via MHC-II, whereas endogenous, cytoplasmic proteins are shuttled to MHC-I molecules for presentation to CD8+ cells. As such, either CD4+ helper T cells or CD8+ cytotoxic T-cell responses are induced. The understanding of this pivotal role played by DCs formed the basis for DC-based vaccination, where a patient's DCs are isolated, modified ex vivo to present tumor antigens and re-administered as a cellular vaccine. DC-based therapeutics, however, suffer from a number of challenges, of which the expensive and lengthy ex vivo procedure for antigen loading and activation of DCs is the most prominent (
      • Santos P.M.
      • Butterfield L.H.
      Dendritic cell-based cancer vaccines.
      ). In this regard, microbubbles have been investigated for direct delivery of tumor antigens to immune cells in vivo.
      • Bioley G.
      • Lassus A.
      • Terrettaz J.
      • Tranquart F.
      • Corthesy B.
      Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria.
      reported that intact microbubbles are rapidly phagocytosed by both murine and human DCs, resulting in rapid and efficient uptake of surface-coupled antigens without the use of ultrasound. Subcutaneous injection of microbubbles loaded with the model antigen ovalbumin (OVA) resulted in the activation of both CD8+ and CD4+ T cells. Effectively, these T-cell responses could partially protect vaccinated mice against an OVA-expressing Listeria infection.
      • Dewitte H.
      • Van Lint S.
      • Heirman C.
      • Thielemans K.
      • De Smedt S.C.
      • Breckpot K.
      • Lentacker I.
      The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy.
      investigated a different approach, making use of messenger RNA (mRNA)-loaded microbubbles combined with ultrasound to transfect DCs. As such, they were able to deliver mRNA encoding both tumor antigens and immunomodulating molecules directly to the cytoplasm of the DCs. As a result, preferential presentation of antigen fragments in MHC-I complexes was ensured, favoring the induction of CD8+ cytotoxic T cells. In a therapeutic vaccination study in mice bearing OVA-expressing tumors, injection of mRNA-sonoporated DCs caused a pronounced slowdown of tumor growth and induced complete tumor regression in 30% of the vaccinated animals. Interestingly, in humans, intradermally injected microbubbles have been used as sentinel lymph node detectors as they can easily drain from peripheral sites to the afferent lymph nodes (
      • Sever A.R.
      • Mills P.
      • Jones S.E.
      • Mali W.
      • Jones P.A.
      Sentinel node identification using microbubbles and contrast-enhanced ultrasonography.
      ,
      • Sever A.R.
      • Mills P.
      • Weeks J.
      • Jones S.E.
      • Fish D.
      • Jones P.A.
      • Mali W.
      Preoperative needle biopsy of sentinel lymph nodes using intradermal microbubbles and contrast-enhanced ultrasound in patients with breast cancer.
      ). As lymph nodes are the primary sites of immune induction, the interaction of microbubbles with intranodal DCs, could be of high value. To this end,
      • Dewitte H.
      • Vanderperren K.
      • Haers H.
      • Stock E.
      • Duchateau L.
      • Hesta M.
      • Saunders J.H.
      • De Smedt S.C.
      • Lentacker I.
      • De S.C.
      Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: Implications for drug delivery.
      found that mRNA-loaded microbubbles were able to rapidly and efficiently migrate to the afferent lymph nodes after intradermal injection in healthy dogs. Unfortunately, further translation of this concept to an in vivo setting is not straightforward, as it prompts the use of less accessible large animal models (e.g., pigs, dogs). Indeed, conversely to what has been reported in humans, lymphatic drainage of subcutaneously injected microbubbles is very limited in the small animal models typically used in pre-clinical research (mice and rats), which is the result of substantial differences in lymphatic physiology.
      Another strategy in cancer immunotherapy is adoptive cell therapy, in which ex vivo manipulated immune effector cells, mainly T cells and natural killer (NK) cells, are employed to generate a robust and selective anticancer immune response (
      • Yee C.
      Adoptive T cell therapy: Points to consider.
      ;
      • Hu W.
      • Wang G.
      • Huang D.
      • Sui M.
      • Xu Y.
      Cancer immunotherapy based on natural killer cells: Current progress and new opportunities.
      ). These strategies have mainly led to successes in hematological malignancies, not only because of the availability of selective target antigens, but also because of the accessibility of the malignant cells (
      • Khalil D.N.
      • Smith E.L.
      • Brentjens R.J.
      • Wolchok J.D.
      The future of cancer treatment: Immunomodulation, CARs and combination immunotherapy.
      ;
      • Yee C.
      Adoptive T cell therapy: Points to consider.
      ). By contrast, in solid tumors, and especially in brain cancers, inadequate homing of cytotoxic T cells or NK cells to the tumor proved to be one of the main reasons for the low success rates, making the degree of tumor infiltration an important factor in disease prognosis (
      • Childs R.W.
      • Carlsten M.
      Therapeutic approaches to enhance natural killer cell cytotoxicity against cancer: The force awakens.
      ;
      • Gras Navarro A.
      • Bjorklund A.T.
      • Chekenya M.
      Therapeutic potential and challenges of natural killer cells in treatment of solid tumors.
      ;
      • Yee C.
      Adoptive T cell therapy: Points to consider.
      ). To address this, focused ultrasound and microbubbles have been used to make tumors more accessible to cellular therapies. The first demonstration of this concept was provided by
      • Alkins R.
      • Burgess A.
      • Ganguly M.
      • Francia G.
      • Kerbel R.
      • Wels W.S.
      • Hynynen K.
      Focused ultrasound delivers targeted immune cells to metastatic brain tumors.
      , who used a xenograft HER-2-expressing breast cancer brain metastasis model to determine whether ultrasound and microbubbles could allow intravenously infused NK cells to cross the BBB. By loading the NK cells with superparamagnetic iron oxide nanoparticles, the accumulation of NK cells in the brain could be tracked and quantified via MRI. An enhanced accumulation of NK cells was found when the cells were injected immediately before BBB disruption. Importantly NK cells retained their activity and ultrasound treatment resulted in a sufficient NK-to-tumor cell ratio to allow effective tumor cell killing (
      • Alkins R.
      • Burgess A.
      • Kerbel R.
      • Wels W.S.
      • Hynynen K.
      Early treatment of HER2-amplified brain tumors with targeted NK-92 cells and focused ultrasound improves survival.
      ). In contrast, very few NK cells reached the tumor site when BBB disruption was absent or performed before NK cell infusion. Although it is not known for certain why timing had such a significant impact on NK extravasation, it is likely that the most effective transfer to the tissue occurs at the time of insonification, and that the barrier is most open during this time (
      • Marty B.
      • Larrat B.
      • Van Landeghem M.
      • Robic C.
      • Robert P.
      • Port M.
      • Le Bihan D.
      • Pernot M.
      • Tanter M.
      • Lethimonnier F.
      • Meriaux S.
      Dynamic study of blood-brain barrier closure after its disruption using ultrasound: A quantitative analysis.
      ). Possible other explanations include the difference in size of the temporal BBB openings or a possible alternation in the expression of specific leukocyte adhesion molecules by the BBB disruption, thus facilitating the translocation of NK cells. Also, for tumors where BBB crossing is not an issue, ultrasound has been used to improve delivery of cellular therapeutics.
      • Sta Maria N.S.
      • Barnes S.R.
      • Weist M.R.
      • Colcher D.
      • Raubitschek A.A.
      • Jacobs R.E.
      Low dose focused ultrasound induces enhanced tumor accumulation of natural killer cells.
      reported enhanced tumor infiltration of adoptively transferred NK cells after treatment with microbubbles and low-dose focused ultrasound. This result was confirmed by
      • Yang C.
      • Du M.
      • Yan F.
      • Chen Z.
      Focused ultrasound improves NK-92 MI cells infiltration into tumors.
      in a more recent publication where the homing of NK cells more than doubled after microbubble injection and ultrasound treatment of an ovarian tumor. Despite the enhanced accumulation, however, the authors did not observe an improved therapeutic effect, which might be owing to the limited number of treatments that were applied or the immunosuppressive tumor microenvironment that counteracts the cytotoxic action of the NK cells.
      There is growing interest in exploring the effect of microbubbles and ultrasound on the tumor microenvironment, as recent work has indicated that BBB disruption with microbubbles and ultrasound may induce sterile inflammation. Although a strong inflammatory response may be detrimental in the case of drug delivery across the BBB, it might be interesting to further study this inflammatory response in solid tumors as it might induce the release of damage-associated molecular patterns (DAMPS) such as heat-shock proteins and inflammatory cytokines. This could shift the balance toward a more inflammatory microenvironment that could promote immunotherapeutic approaches. As reported by
      • Liu H.L.
      • Hsieh H.Y.
      • Lu L.A.
      • Kang C.W.
      • Wu M.F.
      • Lin C.Y.
      Low-pressure pulsed focused ultrasound with microbubbles promotes an anticancer immunological response.
      exposure of a CT26 colon carcinoma xenograft to microbubbles and low-pressure pulsed ultrasound increased cytokine release and triggered lymphocyte infiltration. Similar data have been reported by
      • Hunt S.J.
      • Gade T.
      • Soulen M.C.
      • Pickup S.
      • Sehgal C.M.
      Antivascular ultrasound therapy: Magnetic resonance imaging validation and activation of the immune response in murine melanoma.
      . In their study, ultrasound treatment caused a complete shutdown of tumor vasculature followed by the expression of hypoxia-inducible factor 1α (HIF-1α), a marker of tumor ischemia and tumor necrosis, as well as increased infiltration of T cells. Similar responses have been reported after thermal and mechanical HIFU treatments of solid tumors (
      • Unga J.
      • Hashida M.
      Ultrasound induced cancer immunotherapy.
      ;
      • Silvestrini M.T.
      • Ingham E.S.
      • Mahakian L.M.
      • Kheirolomoom A.
      • Liu Y.
      • Fite B.Z.
      • Tam S.M.
      • Tucci S.T.
      • Watson K.D.
      • Wong A.W.
      • Monjazeb A.M.
      • Hubbard N.E.
      • Murphy W.J.
      • Borowsky A.D.
      • Ferrara K.W.
      Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols.
      ). A detailed review of ablative ultrasound therapies is, however, out of the scope of this review.
      At present, the most successful form of immunotherapy is the administration of monoclonal antibodies to inhibit regulatory immune checkpoints that block T-cell action. Examples are cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), which act as brakes on the immune system. Blocking the effect of these brakes can revive and support the function of immune effector cells. Despite the numerous successes achieved with checkpoint inhibitors, responses have been quite heterogeneous as the success of checkpoint inhibition therapy depends largely on the presence of intra-tumoral effector T cells (
      • Weber J.S.
      Biomarkers for checkpoint inhibition.
      ). This motivated
      • Bulner S.
      • Prodeus A.
      • Gariepy J.
      • Hynynen K.
      • Goertz D.E.
      Enhancing checkpoint inhibitor therapy with ultrasound stimulated microbubbles.
      to explore the synergy of microbubble and ultrasound treatment with PD-L1 checkpoint inhibition therapy in mice. Tumors in the treatment group that received the combination of microbubble and ultrasound treatment with checkpoint inhibition were significantly smaller than tumors in the monotherapy groups. One mouse exhibited complete tumor regression and remained tumor free upon rechallenge, indicative of an adaptive immune response.
      Overall, the number of studies that have investigated the impact of microbubble and ultrasound treatment on immunotherapy is limited, making this a rather unexplored research area. It is obvious that more in-depth research is warranted to improve our understanding on how (various types of) immunotherapy might benefit from (various types of) ultrasound treatment.

      BBB and blood–spinal cord barrier opening

      The barriers of the central nervous system (CNS), the BBB and blood–spinal cord barrier (BSCB), greatly limit drug-based treatment of CNS disorders. These barriers help to regulate the specialized CNS environment by limiting the passage of most therapeutically relevant molecules (
      • Pardridge W.M.
      The blood-brain barrier: Bottleneck in brain drug development.
      ). Although several methods have been proposed to circumvent the BBB and BSCB, including chemical disruption and the development of molecules engineered to capitalize on receptor-mediated transport (so-called Trojan horse molecules), the use of ultrasound in combination with microbubbles (
      • Hynynen K.
      • McDannold N.
      • Vykhodtseva N.
      • Jolesz F.A.
      Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.
      ) or droplets (
      • Wu S.Y.
      • Fix S.M.
      • Arena C.B.
      • Chen C.C.
      • Zheng W.
      • Olumolade O.O.
      • Papadopoulou V.
      • Novell A.
      • Dayton P.A.
      • Konofagou E.E.
      Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: Vaporization efficiency dictates large molecular delivery.
      ) to transiently modulate these barriers has come to the forefront in recent years because of the targeted nature of this approach and its ability to facilitate delivery of a wide range of currently available therapeutics. First demonstrated in 2001 (
      • Hynynen K.
      • McDannold N.
      • Vykhodtseva N.
      • Jolesz F.A.
      Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.
      ), ultrasound-mediated BBB opening has been the topic of several hundred original research articles in the last two decades and, in recent years, has made headlines for groundbreaking clinical trials targeting brain tumors and Alzheimer's disease as described later under Clinical Studies.

      Mechanisms, bio-effects and tolerability

      Ultrasound in combination with microbubbles can produce permeability changes in the BBB via both enhanced paracellular and transcellular transport (
      • Sheikov N.
      • McDannold N.
      • Vykhodtseva N.
      • Jolesz F.
      • Hynynen K.
      Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles.
      ,
      • Sheikov N.
      • McDannold N.
      • Jolesz F.
      • Zhang Y.Z.
      • Tam K.
      • Hynynen K.
      Brain arterioles show more active vesicular transport of blood-borne tracer molecules than capillaries and venules after focused ultrasound-evoked opening of the blood-brain barrier.
      ). Reduction and reorganization of tight junction proteins (
      • Sheikov N.
      • McDannold N.
      • Sharma S.
      • Hynynen K.
      Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium.
      ) and upregulation of active transport protein caveolin-1 (
      • Deng J.
      • Huang Q.
      • Wang F.
      • Liu Y.
      • Wang Z.
      • Zhang Q.
      • Lei B.
      • Cheng Y.
      The role of caveolin-1 in blood-brain barrier disruption induced by focused ultrasound combined with microbubbles.
      ) have been reported. Although the exact physical mechanisms driving these changes are not known, there are several factors that are hypothesized to contribute to these effects, including direct tensile stresses caused by the expansion and contraction of the bubbles in the lumen, as well as shear stresses at the vessel wall arising from acoustic microstreaming. Recent studies have also investigated the suppression of efflux transporters after ultrasound exposure with microbubbles. A reduction in P-glycoprotein expression (
      • Cho H.
      • Lee H.Y.
      • Han M.
      • Choi J.R.
      • Ahn S.
      • Lee T.
      • Chang Y.
      • Park J.
      Localized down-regulation of P-glycoprotein by focused ultrasound and microbubbles induced blood-brain barrier disruption in rat brain.
      ;
      • Aryal M.
      • Fischer K.
      • Gentile C.
      • Gitto S.
      • Zhang Y.Z.
      • McDannold N.
      Effects on P-glycoprotein expression after blood-brain barrier disruption using focused ultrasound and microbubbles.
      ) and BBB transporter gene expression (
      • McMahon D.
      • Mah E.
      • Hynynen K.
      Angiogenic response of rat hippocampal vasculature to focused ultrasound-mediated increases in blood-brain barrier permeability.
      ) has been observed by multiple groups. One study found that P-glycoprotein expression was suppressed for more than 48 h after treatment with ultrasound and microbubbles (
      • Aryal M.
      • Fischer K.
      • Gentile C.
      • Gitto S.
      • Zhang Y.Z.
      • McDannold N.
      Effects on P-glycoprotein expression after blood-brain barrier disruption using focused ultrasound and microbubbles.
      ). However, the degree of inhibition of efflux transporters as a result of ultrasound with microbubbles may be insufficient to prevent efflux of some therapeutics (
      • Goutal S.
      • Gerstenmayer M.
      • Auvity S.
      • Caillé F.
      • Mériaux S.
      • Buvat I.
      • Larrat B.
      • Tournier N.
      Physical blood-brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib.
      ), and thus this mechanism requires further study.
      Many studies have documented enhanced CNS tumor response after ultrasound and microbubble-mediated delivery of drugs across the blood–tumor barrier in rodent models. Improved survival has been observed in both primary (
      • Chen P.Y.
      • Liu H.L.
      • Hua M.Y.
      • Yang H.W.
      • Huang C.Y.
      • Chu P.C.
      • Lyu L.A.
      • Tseng I.C.
      • Feng L.Y.
      • Tsai H.C.
      • Chen S.M.
      • Lu Y.J.
      • Wang J.J.
      • Yen T.C.
      • Ma Y.H.
      • Wu T.
      • Chen J.P.
      • Chuang J.I.
      • Shin J.W.
      • Hsueh C.
      • Wei K.C.
      Novel magnetic/ultrasound focusing system enhances nanoparticle drug delivery for glioma treatment.
      ;
      • Aryal M.
      • Vykhodtseva N.
      • Zhang Y.Z.
      • Park J.
      • McDannold N.
      Multiple treatments with liposomal doxorubicin and ultrasound-induced disruption of blood-tumor and blood-brain barriers improve outcomes in a rat glioma model.
      ) and metastatic (
      • Park E.J.
      • Zhang Y.Z.
      • Vykhodtseva N.
      • McDannold N.
      Ultrasound-mediated blood-brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model.
      ;
      • Alkins R.
      • Burgess A.
      • Kerbel R.
      • Wels W.S.
      • Hynynen K.
      Early treatment of HER2-amplified brain tumors with targeted NK-92 cells and focused ultrasound improves survival.
      ) tumor models.
      Beyond simply enhancing drug accumulation in the CNS, several positive bio-effects of ultrasound and microbubble-induced BBB opening have been reported. In rodent models of Alzheimer's disease, numerous positive effects have been discovered in the absence of exogenous therapeutics. These effects include a reduction in amyloid-β plaque load (
      • Jordão J.F.
      • Thévenot E.
      • Markham-Coultes K.
      • Scarcelli T.
      • Weng Y.Q.
      • Xhima K.
      • O'Reilly M.
      • Huang Y.
      • McLaurin J.
      • Hynynen K.
      • Aubert I.
      Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound.
      ;
      • Burgess A.
      • Dubey S.
      • Yeung S.
      • Hough O.
      • Eterman N.
      • Aubert I.
      • Hynynen K.
      Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior.
      ;
      • Leinenga G.
      • Götz J.
      Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model.
      ;
      • Poon C.T.
      • Shah K.
      • Lin C.
      • Tse R.
      • Kim K.K.
      • Mooney S.
      • Aubert I.
      • Stefanovic B.
      • Hynynen K.
      Time course of focused ultrasound effects on β-amyloid plaque pathology in the TgCRND8 mouse model of Alzheimer's disease.
      ), reduction in tau pathology (
      • Pandit R.
      • Leinenga G.
      • Götz J.
      Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.
      ) and improvements in spatial memory (
      • Burgess A.
      • Dubey S.
      • Yeung S.
      • Hough O.
      • Eterman N.
      • Aubert I.
      • Hynynen K.
      Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior.
      ;
      • Leinenga G.
      • Götz J.
      Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model.
      ). Two-photon microscopy has revealed that amyloid-β plaque size is reduced in transgenic mice for up to 2 wk after ultrasound and microbubble treatment (
      • Poon C.T.
      • Shah K.
      • Lin C.
      • Tse R.
      • Kim K.K.
      • Mooney S.
      • Aubert I.
      • Stefanovic B.
      • Hynynen K.
      Time course of focused ultrasound effects on β-amyloid plaque pathology in the TgCRND8 mouse model of Alzheimer's disease.