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Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements

      Abstract

      Initial reports from the 1960s describing the observations of ultrasound contrast enhancement by tiny gaseous bubbles during echocardiographic examinations prompted the development of the first ultrasound contrast agent in the 1980s. Current commercial contrast agents for echography, such as Definity, Optison, Sonazoid and SonoVue, have proven to be successful in a variety of on- and off-label clinical indications. Whereas contrast-specific technology has seen dramatic progress after the introduction of the first approved agents in the 1990s, successful clinical translation of new developments has been limited during the same period, while understanding of microbubble physical, chemical and biologic behavior has improved substantially. It is expected that for a successful development of future opportunities, such as ultrasound molecular imaging and therapeutic applications using microbubbles, new creative developments in microbubble engineering and production dedicated to further optimizing microbubble performance are required, and that they cannot rely on bubble technology developed more than 3 decades ago.

      Key Words

      Introduction: A Review of the Past

      A Cloud of Echoes

      The diagnostic applications of ultrasound imaging have been expanded enormously during the last few decades. With the introduction of ultrasound contrast agents (UCA), new meaningful physiologic and pathologic information is provided, and perfusion imaging of myocardial or tumor tissue has now become available for routine clinical decision making. The first observations of the ultrasonic contrast effect date from the mid-1960s to work by Joyner (
      • Gramiak R.
      • Shah P.M.
      Echocardiography of the aortic root.
      ). Indeed, contrast echocardiography started when it was noted by
      • Gramiak R.
      • Shah P.M.
      • Kramer D.H.
      Ultrasound cardiography: Contrast studies in anatomy and function.
      that the intracardiac injection of indocyanine green dye, a frequently used substance for measuring blood flow, produced a “cloud of echoes” on the M-mode echocardiogram (
      • Gramiak R.
      • Shah P.M.
      Echocardiography of the aortic root.
      ). In fact, it was shown that the injection of almost any liquid through a small-bore needle or catheter would produce this contrast effect (
      • Kremkau F.W.
      • Gramiak R.
      • Carstensen E.L.
      • Shah P.M.
      • Kramer D.H.
      Ultrasonic detection of cavitation at catheter tips.
      ;
      • Kremkau F.W.
      • Gramiak R.
      • Carstensen E.L.
      • Shah P.M.
      • Kramer D.H.
      Ultrasonic detection of cavitation at catheter tips.
      ).
      The effect was referred to as a cloud of echoes although investigators speculated that the phenomenon was owing to the presence of tiny gas bubbles suspended in the liquid (
      • Bove A.A.
      • Adams D.F.
      • Hugh A.E.
      • Lynch P.R.
      Cavitation at catheter tips. A possible cause of air embolism.
      ;
      • Ziskin M.C.
      • Bonakdarpour A.
      • Weinstein D.P.
      • Lynch P.R.
      Contrast agents for diagnostic ultrasound.
      ;
      • Barrera J.G.
      • Fulkerson P.K.
      • Rittgers S.E.
      • Nerem R.
      The nature of contrast echocardiographic “targets”.
      ). Nevertheless, this correct hypothesis was still challenged, and others suggested that particulate matter caused the ultrasound contrast (
      • Schuchman H.
      • Feigenbaum H.
      • Dillon J.C.
      • Chang S.
      Intracavitary echoes in patients with mitral prosthetic valves.
      ).
      • Meltzer R.
      • Tickner G.
      • Sahines T.
      • Popp R.L.
      The source of ultrasonic contrast effect.
      provided evidence on this subject and demonstrated, by examining fluids before and after hand agitation employing two syringes connected by a three-way stopcock, that the microbubbles used for peripheral contrast echocardiography were formed during this agitation and thus were already present in the injectant rather than being formed at the catheter tip during injection.
      Although free gas bubbles could be generated in any liquid, it was observed that in indocyanine green dye and gelatin microbubble persistence increased, improving contrast enhancement (
      • Meltzer R.
      • Tickner G.
      • Sahines T.
      • Popp R.L.
      The source of ultrasonic contrast effect.
      ;
      • Carroll B.A.
      • Turner R.J.
      • Tickner E.G.
      • Boyle D.B.
      • Young S.W.
      Gelatin encapsulated nitrogen microbubbles as ultrasonic contrast agents.
      ). It was concluded that these liquids act as surfactants, decreasing the surface tension to values lower than that of a clean saline-air interface, thereby stabilizing the free gas bubbles against rapid dissolution. Moreover, the surfactants prevented bubbles from coalescing, minimizing the creation of large and potentially dangerous bubble sizes (
      • Meltzer R.
      • Tickner G.
      • Sahines T.
      • Popp R.L.
      The source of ultrasonic contrast effect.
      ). Nevertheless, free gas bubbles produced by hand agitation of saline are sometimes still being used in echocardiography for detecting intracardiac shunts.
      In the period between 1970–1980, the field of contrast echography evolved further and mainly focused on a wide variety of applications in cardiology such as identification of cardiac structures and cavity dimensions (
      • Roelandt J.
      Contrast echocardiography.
      ), detection of intracardiac shunts (
      • Valdes-Cruz L.M.
      • Sahn D.J.
      Ultrasonic contrast studies for the detection of cardiac shunts.
      ), visualization of blood flow in M-mode, detection of valvular regurgitation (
      • Kerber R.E.
      • Kioschos J.M.
      • Lauer R.M.
      Use of an ultrasonic contrast method in the diagnosis of valvular regurgitation and intracardiac shunts.
      ;
      • Reid C.L.
      • Kawanishi D.T.
      • McKay C.R.
      Accuracy of evaluation of the presence and severity of aortic and mitral regurgitation by contrast 2-dimensional echocardiography.
      ), analysis of complex congenital heart disease and cardiac output determination by indicator dilution curves (
      Contrast echocardiography.
      ). However, the full potential of contrast echography could still not be explored because of inherent shortcomings of free gaseous microbubbles, such as very short lifetime and low persistence, indeterminate size and inability to pass through the lung circulation after an intravenous injection. Nevertheless, the expectation and confidence in this new modality was very strong, as is illustrated by the following passage from Contrast Echocardiography edited by
      Contrast echocardiography.
      :
      • The future of contrast echocardiography is almost unlimited. As this book indicates, there is a vast amount of interest and research currently being done with regards to contrast echocardiography. Probably the most exciting aspect of this research is the development of new contrast producing agents. It is going to be exciting to see how these various agents develop. Hopefully, one or more of these new agents will be able to traverse the capillaries so that one can visualize the left side of the heart with a peripheral venous injection.
      This passage also illustrates the awareness of the limitations at that time, and it took more than a decade to develop the first commercial contrast agent for imaging the left ventricle (LV) of the heart.

      Development of the “ideal” UCA

      From 1980, extensive research was performed in order to make contrast echocardiography an established diagnostic technique (
      • Senior R.
      • Becher H.
      • Monaghan M.
      • Agati L.
      • Zamorano J.
      • Vanoverschelde J.L.
      • Nihoyannopoulos P.
      • Edvardsen T.
      • Lancellotti P.
      Clinical practice of contrast echocardiography: Recommendation by the European Association of Cardiovascular Imaging (EACVI) 2017.
      ). In 1989,
      • Ophir J.
      • Parker K.J.
      Contrast agents in diagnostic ultrasound.
      summarized the use of UCA in medical imaging. Five types of agents with different physical properties were classified: free gas bubbles, encapsulated gas bubbles, colloidal suspensions, emulsions and aqueous solutions. In those days, it was still a main challenge to produce the “ideal” contrast agent, which would meet the following criteria:
      • Distribution of the agent within the heart chamber or myocardium representative of local blood flow;
      • Stability of the agent to persist during an imaging examination after an intravenous injection;
      • Consisting of microbubbles smaller than 8 µm in diameter (smaller than red blood cells) enabling passage through the pulmonary system and the smallest capillaries of the body;
      • Physiologically inert, excellent safety profile;
      • Echogenic, strong and controlled acoustic interaction.

      Stabilized microbubbles

      The challenge of producing stable encapsulated microbubbles surviving passage through the heart and the pulmonary capillary network was first resolved in 1984 (
      • Feinstein S.B.
      • Shah P.M.
      • Bing R.J.
      • Meerbaum S.
      • Corday E.
      • Chang B.L.
      • Santillan G.
      • Fujibayashi Y.
      Microbubble dynamics visualized in the intact capillary circulation.
      ), when microbubbles were produced by cavitation after introducing the tip of a sonicator horn into a solution of human serum albumin. These microbubbles could be visualized in the left heart after a peripheral venous injection. During the 1990s, research on gaseous microbubbles as an UCA became very active, and numerous manufacturers started to develop new microbubble-based contrast agents that met most of the criteria listed above. Several technologies for stabilizing the microbubbles were investigated. Thin shells made of protein, polymer or phospholipids were used to reduce surface tension and stabilize the gas core against rapid dissolution. However, the first-generation agents still suffered from limited stability and very short circulation time because of the high solubility of air in water. Persistence during circulation was dramatically improved by replacing air by perfluorinated gases with a low solubility in water, such as sulphur hexafluoride (
      • Schneider M.
      • Arditi M.
      • Barrau M.B.
      • Brochot J.
      • Broillet A.
      • Ventrone R.
      • Yan F.
      BR1: A new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles.
      ), perfluoropropane (
      • Unger E.
      • Shen D.
      • Fritz T.
      • Kulik B.
      • Lund P.
      • Wu G.L.
      • Yellowhair D.
      • Ramaswami R.
      • Matsunage T.
      Gas-filled lipid bilayers as ultrasound contrast agents.
      ) or perfluorobutane (
      • Schneider M.
      • Broillet A.
      • Bussat P.
      • Giessinger N.
      • Puginier J.
      • Ventrone R.
      • Yan F.
      Gray-scale liver enhancement in VX2 tumor-bearing rabbits using BR14, a new ultrasonographic contrast agent.
      ;
      • 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.
      ), resulting in a persistence of the agent in the blood circulation sufficient for clinical use. At the end of the decade, more than 15 different agents were at various stages of development. Table 1 summarizes a non-exhaustive list of agents in development for intravenous application at that time (
      • Kasprzak J.D.
      • Ten Cate F.J.
      New ultrasound contrast agents for left ventricular and myocardial opacification.
      ;
      • Becher H.
      • Burns P.N.
      Handbook of contrast echocardiography: Left ventricular function and myocardial perfusion.
      ;
      • Stride E.
      • Saffari N.
      Microbubble ultrasound contrast agents: A review.
      ;
      • Quaia E.
      Classification and safety of microbubble-based contrast agents.
      ;
      • Faez T.
      • Emmer M.
      • Kooiman K.
      • Versluis M.
      • van der Steen A.
      • de Jong N.
      20 years of ultrasound contrast agent modeling.
      ;
      • Chang E.H.
      An Introduction to Contrast-Enhanced Ultrasound for Nephrologists.
      ).
      Table 1Microbubble-based ultrasound contrast agents for intravenous injection that reached different stages of development during the period between 1990 and 2000.
      Trademark nameCode nameManufacturerFormulations: shell/filling gas
      AlbunexMallinckrodt PharmaceuticalsHuman albumin/air
      BispherePB127Point BiomedicalPolymer bilayer–albumin/air
      Definity/LuminityMRX-115/DMP-115Bristol-Myers SquibbPhospholipids/perfluoropropane
      EchogenQW3600Sonus PharmaceuticalsSurfactant/dodecafluoropentane
      EchovistSH U454Schering AGGalactose/air
      FilmixCavconLipid/air
      Imavist (Imagent)AFO150 (AFO145)Imcor (Alliance) PharmaceuticalsPhospholipids/perfluorohexane-air
      LevovistSH U508 ASchering AGGalactose-palmitic acid/air
      MyomapAIP 201Quadrant HealthcareRecombinant albumin/air
      OptisonFS069Amersham Health Inc.Protein-type A/perfluoropropane
      QuantisonAIP101Quadrant HealthcareRecombinant albumin/air
      SonavistSH U563 ASchering AGPolymer/air
      SonazoidNC100100Amersham Health Inc.Lipid/perfluorobutane
      SonoGenQW7437Sonus PharmaceuticalsSurfactant/dodecafluoropentane
      SonoVue/LumasonBR1Bracco Imaging S.p.A.,Phospholipids/sulphur hexafluoride
      AI-700Acusphere Inc.Polymer/perflourobutane
      BR14Bracco Diagnostics Inc.Phospholipids/perfluorobutane
      BR38Bracco Diagnostics Inc.Phospholipids/perfluorobutane-nitrogen
      Adapted from
      • Kasprzak J.D.
      • Ten Cate F.J.
      New ultrasound contrast agents for left ventricular and myocardial opacification.
      ;
      • Becher H.
      • Burns P.N.
      Handbook of contrast echocardiography: Left ventricular function and myocardial perfusion.
      ;
      • Stride E.
      • Saffari N.
      Microbubble ultrasound contrast agents: A review.
      ;
      • Quaia E.
      Classification and safety of microbubble-based contrast agents.
      ;
      • Faez T.
      • Emmer M.
      • Kooiman K.
      • Versluis M.
      • van der Steen A.
      • de Jong N.
      20 years of ultrasound contrast agent modeling.
      ;
      • Chang E.H.
      An Introduction to Contrast-Enhanced Ultrasound for Nephrologists.
      .
      The introduction of the first microbubbles satisfying most criteria required for an intravenous UCA also prompted tremendous research efforts by clinicians, scientists and ultrasound equipment manufacturers to describe the physical phenomena and to translate the knowledge into clinical applications. For example, theoretical expressions for cavitation (
      • Atchley A.A.
      • Crum L.A.
      Acoustic cavitation and bubble dynamics.
      ), the mechanism used for describing the cloud of echoes observed in the beginning on M-mode echocardiograms, proved to be very useful for understanding the interaction between ultrasound waves and gaseous microbubbles. Particularly, the high compressibility of the gas core appearde to be important, since it results in frequency-dependent volume pulsations with a pronounced maximum at the resonance frequency, with the resonance frequency being inversely proportional to the microbubble size (
      • Minnaert M.
      On musical air-bubbles and the sound of running water.
      ;
      • Medwin H.
      Counting bubbles acoustically: a review.
      ). Fortunately, the range of microbubble sizes appropriate for clinical use have resonance frequencies on the order of 1–15 MHz, right in the frequency range commonly used in diagnostic ultrasound (
      • Mulvana H.
      • Browning R.J.
      • Luan Y.
      • de Jong N.
      • Tang M.X.
      • Eckersley R.J.
      • Stride E.
      Characterization of contrast agent microbubbles for ultrasound imaging and therapy research.
      ). Moreover, a resonating bubble behaves as a source of sound rather than as a passive scatterer, yielding an enhancement of particularly non-linear echo signals.

      Non-linear microbubble behavior

      The non-linear echoes produced by gaseous microbubbles stem from intrinsic non-linearities in the Rayleigh-Plesset equation. However, for stabilized microbubbles, the presence of the encapsulating shell substantially affects the volumetric microbubble oscillation amplitude, and the viscoelastic properties of the shell strongly alter its resonance behavior. It has been observed that because of the presence of the shell, encapsulated gas bubbles can be up to 20 times more rigid than free gas bubbles, substantially increasing the resonance frequency. Moreover, because of shell viscosity, bubble oscillations are heavily damped, attenuating the propagating ultrasound wave and thus limiting acoustic penetration depth (
      • de Jong N.
      • Hoff L.
      Ultrasound scattering properties of Albunex microspheres.
      ;
      • Church C.C.
      The effects of an elastic solid surface layer on the radial pulsations of gas bubbles.
      ;
      • Sontum P.C.
      • Østensen J.
      • Dyrstad K.
      • Hoff L.
      Acoustic properties of NC100100 and their relationship with the microsphere size distribution.
      ;
      • Hoff L.
      • Sontum P.C.
      • Hovem J.M.
      Oscillations of polymeric microbubbles: Effect of the encapsulating shell.
      ;
      • Sarkar K.
      • Shi W.T.
      • Chatterjee D.
      • Forsberg F.
      Characterization of ultrasound contrast microbubbles using in vitro experiments and viscous and viscoelastic interface models for encapsulation.
      ). Since early- to mid-2000, Rayleigh-Plesset–type models including non-linear shell elasticity and viscosity terms were developed (
      • Marmottant P.
      • VanderMeer S.
      • Emmer M.
      • Versluis M.
      • de Jong N.
      • Hilgenfeldtb S.
      • Lohse D.
      A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture.
      ;
      • Stride E.
      The influence of surface adsorption on microbubble dynamics.
      ;
      • Tsiglifis K.
      • Pelekasis N.A.
      Nonlinear radial oscillations of encapsulated microbubbles subject to ultrasound: The effect of membrane constitutive law.
      ;
      • Paul S.
      • Katiyar A.
      • Sarkar K.
      • Chatterjee D.
      • Shi W.T.
      • Forsberg F.
      Material characterization of the encapsulation of an ultrasound contrast microbubble and its subharmonic response: Strain-softening interfacial elasticity model.
      ;
      • Katiyar A.
      • Sarkar K.
      Excitation threshold for subharmonic generation from contrast microbubbles.
      ), predicting the non-linear dynamics of an oscillating microbubble particularly owing to the presence of the stabilizing shell. Excellent reviews on contrast agent modelling are given by
      • Doinikov A.A.
      • Bouakaz A.
      Review of shell models for contrast agent microbubbles.
      and
      • Faez T.
      • Emmer M.
      • Kooiman K.
      • Versluis M.
      • van der Steen A.
      • de Jong N.
      20 years of ultrasound contrast agent modeling.
      .
      The non-linear dynamics of stabilized oscillating microbubbles includes “compression only behavior,” an asymmetric oscillation where a microbubble compresses more than it expands, which was observed experimentally by
      • de Jong N.
      • Emmer M.
      • Chin C.T.
      • Bouakaz A.
      • Mastik F.
      • Lohse D.
      • Versluis M.
      “Compression-only” behavior of phospholipid-coated contrast bubbles.
      using high-speed imaging. Compression-only results from the non-linear surface tension of an encapsulated microbubble with an equilibrium surface tension close to zero. During compression, the microbubble reaches the tensionless buckling state, whereas volumetric expansions are counteracted by the finite shell elasticity once the bubble reaches the elastic state (
      • Marmottant P.
      • VanderMeer S.
      • Emmer M.
      • Versluis M.
      • de Jong N.
      • Hilgenfeldtb S.
      • Lohse D.
      A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture.
      ,
      • Marmottant P.
      • Bouakaz A.
      • de Jong N.
      • Quilliet C.
      Buckling resistance of solid shell bubbles under ultrasound.
      ). Another observation resulting from shell non-linearity, initially described as thresholding (
      • Emmer M.
      • van Wamel A.
      • Goertz D.E.
      • de Jong N.
      The onset of microbubble vibration.
      ), appeared to be related to a downshift in the resonance frequency with increasing acoustic pressure at low amplitudes (
      • Overvelde M.
      • Garbin V.
      • Sijl J.
      • Dollet B.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Nonlinear shell behavior of phospholipid-coated microbubbles.
      ). Others related this behavior to strain softening of the shell (
      • Tsiglifis K.
      • Pelekasis N.A.
      Nonlinear radial oscillations of encapsulated microbubbles subject to ultrasound: The effect of membrane constitutive law.
      ;
      • Paul S.
      • Katiyar A.
      • Sarkar K.
      • Chatterjee D.
      • Shi W.T.
      • Forsberg F.
      Material characterization of the encapsulation of an ultrasound contrast microbubble and its subharmonic response: Strain-softening interfacial elasticity model.
      ). In fact, the downshift in resonance frequency results from an effective averaging of the shell stiffness over elastic and non-elastic regions, where the elastic region is limited to a relatively narrow microbubble surface area range around the equilibrium bubble area. Thus, the non-linear response of encapsulated microbubbles already appears at very low acoustic pressure amplitudes (e.g., lower than 50 kPa). Higher acoustic pressure amplitudes can result in microbubble destruction and fragmentation. When higher acoustic pressures are applied (e.g., 100–150 kPa for soft-shelled microbubbles and higher than 300 kPa for hard-shelled microbubbles), the shell ruptures transiently and releases free gas bubbles (
      • Frinking P.
      • de Jong N.
      • Cespedes I.
      Scattering properties of encapsulated gas bubbles at high ultrasound pressures.
      ), which can rapidly dissolve in the surrounding liquid. This can generate an abrupt increase of the non-linear harmonics in the backscattered signal, which is a very sensitive way for detecting microbubbles during contrast-enhanced imaging.

      Contrast-specific imaging techniques

      In parallel with the developments of UCA, and because of the improved understanding of non-linear microbubble behavior, specific microbubble imaging modes were developed, and these are currently implemented in most clinical ultrasound systems (
      • Burns P.N.
      • Powers J.E.
      • Hope Simpson D.
      • Uhlendorf V.
      • Fritzsch T.
      Harmonic imaging: Principles and preliminary results.
      ;
      • Wei K.
      • Jayaweer A.R.
      • Firoozan S.
      • Linka A.
      • Skyba D.M.
      • Kaul S.
      Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion.
      ; Hope
      • Simpson D.H.
      • Chin C.T.
      • Burns P.N.
      Pulse inversion Doppler: A new method for detecting nonlinear echoes from microbubble contrast agents.
      ). The contrast-specific imaging modes take advantage of non-linear acoustic properties of gas bubbles very different from those of tissue (
      • Frinking P.
      • Bouakaz A.
      • Kirkhorn J.
      • Ten Cate F.J.
      • de Jong N.
      Ultrasound contrast imaging: Current and new potential methods.
      ;
      • Rafter P.
      • Phillips P.
      • Vannan M.A.
      Imaging technologies and techniques.
      ). Particularly, the non-linear shell behavior explains the success of low mechanical index (MI) (<0.1) contrast-specific imaging mostly used for real-time perfusion and intra-cavitary assessments. Additionally, several diagnostic imaging techniques and/or quantification methods (e.g., harmonic power Doppler, destruction-replenishment imaging) are based on the unique and very sensitive property of microbubble destruction when short pulse excitations at higher MI are applied (
      • Blomley M.J.
      • Albrecht T.
      • Cosgrove D.O.
      • Patel N.
      • Jayaram V.
      • Butler-Barnes J.
      • Eckersley R.J.
      • Bauer A.
      • Schlief R.
      Improved imaging of liver metastases with stimulated acoustic emission in the late phase of enhancement with the US contrast agent SH U 508A: Early experience.
      ;
      • Albrecht T.
      • Blomley M.J.
      • Burns P.N.
      • Wilson S.
      • Harvey C.J.
      • Leen E.
      • Claudon M.
      • Calliada F.
      • Correas J.M.
      • LaFortune M.
      • Campani R.
      • Hoffmann C.W.
      • Cosgrove D.O.
      • LeFevre F.
      Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508: A multicenter study.
      ). Integration of these bubble-specific signatures in combination with excellent tissue suppression algorithms in imaging systems resulted eventually in broad clinical acceptance of contrast-enhanced ultrasound imaging as a diagnostic mode, particularly outside cardiology, with equivalent or even superior clinical performance compared with other contrast-enhanced modalities such as magnetic resonance imaging (MRI) and computed tomography (CT) (
      • Lu M.D.
      • Yu X.L.
      • Li A.H.
      • Jiang T.A.
      • Chen M.H.
      • Zhao B.Z.
      • Zhou X.D.
      • Wang J.R.
      Comparison of contrast enhanced ultrasound and contrast enhanced CT or MRI in monitoring percutaneous thermal ablation procedure in patients with hepatocellular carcinoma: A multi-center study in China.
      ;
      • Meloni M.F.
      • Bertolotto M.
      • Alberzoni C.
      • Lazzaroni S.
      • Filice C.
      • Livraghi T.
      • Ferraioli G.
      Follow-up after percutaneous radiofrequency ablation of renal cell carcinoma: Contrast-enhanced sonography versus contrast-enhanced CT or MRI.
      ;
      • Sidhu P.S.
      • Cantisani V.
      • Dietrich C.F.
      • Gilja O.H.
      • Saftoiu A.
      • Bartels E.
      • Bertolotto M.
      • Calliada F.
      • Clevert D.A.
      • Cosgrove D.
      • Deganello A.
      • D'Onofrio M.
      • Drudi F.M.
      • Freeman S.
      • Harvey C.
      • Jenssen C.
      • Jung E.M.
      • Klauser A.S.
      • Lassau N.
      • Meloni M.F.
      • Leen E.
      • Nicolau C.
      • Nolsoe C.
      • Piscaglia F.
      • Prada F.
      • Prosch H.
      • Radzina M.
      • Savelli L.
      • Weskott H.P.
      • Wijkstra H.
      The EFSUMB guidelines and recommendations for the clinical practice of contrast-enhanced ultrasound (CEUS) in non-hepatic applications: Update 2017 (long version).
      ).

      The present: Commercially available contrast agents

      Most of the agents initially developed for intravenous use are listed in Table 1, and from those reaching clinical approval during the last 3 decades (Table 2), only four are currently marketed and used in routine clinical practice. Three of these agents are phospholipid-shelled agents (SonoVue, Definity, Sonazoid) and one is an albumin-shelled agent (Optison). The formulations, physico-chemical properties, clinical applications and new development of these agents are described in further detail below and summarized in Tables 3 and 4.
      Table 2Clinically approved ultrasound contrast agents.
      NameFirst approved for clinical useShell materialGasApplication (examples)Producer/distributorCountries
      Optison1998Cross-linked serum albuminOctafluoropropaneLeft ventricular opacification, endocardial border delineation, DopplerGE healthcare, Buckinghamshire, UKUSA, Europe
      Sonazoid2006Hydrogenated egg yolk phosphatidyl serine (HEPS)PerfluorobutaneMyocardial perfusion, liver imagingGE healthcare, Buckinghamshire, UK/ Daiichi Sankyo, Tokyo, JapanJapan, South Korea, Norway, Taiwan, China
      Lumason/SonoVue2001/2014PhospholipidSulphur hexafluorideLeft ventricular opacification, microvascular enhancement (liver and breast lesion detection)Bracco Diagnostics Inc., Monroe Township, NJ, USA/Bracco Imaging S.p.A., Colleretto Giacosa, ItalyUSA, Europe, China, Brazil
      Definity/Luminity2001/2006PhospholipidOctafluoropropaneEchocardiography, liver/kidney imaging (Canada)Lantheus Medical Imaging Inc, North Billerica, MA, USANorth America, Europe
      Imagent/Imavist2002, withdrawnPhospholipidPerfluorohexane, NitrogenEchocardiography, heart perfusion, tumor/blood flow anomaliesSchering AG, Berlin, DEUSA
      Echovist1991, withdrawnGalactose microparticlesAirRight heart imagingSchering AG, Berlin, DEGermany, UK
      Levovist1995, withdrawnGalactose microparticles, palmitic acidAirWhole heart imaging, Doppler imagingSchering AG, Berlin, DECanada, Europe, China, Japan
      Albunex1993, withdrawnSonicated serum albuminAirTranspulmonary imagingMolecular Biosystems Inc., San Diego, CA, USAJapan, USA
      Adapted from
      • Paefgen V.
      • Doleschel D.
      • Kiessling F.
      Evolution of contrast agents for ultrasound imaging and ultrasound-mediated drug delivery.
      .
      Table 3Clinical ultrasound contrast agents currently commercially available.
      SonoVue/LumasonDefinity/LuminityOptisonSonazoid
      ManufacturerBracco Imaging S.p.A.Lantheus Medical Imaging IncGE HealthcareGE healthcare/Daiichi Sankyo
      Approval indication(s)LVO/EBD, breast,
      Only in certain countries. Adapted from Chang 2018.
      liver,
      Only in certain countries. Adapted from Chang 2018.
      vascular,
      Only in certain countries. Adapted from Chang 2018.
      urinary tract
      LVO/EBD, breast,
      Only in certain countries. Adapted from Chang 2018.
      liver,
      Only in certain countries. Adapted from Chang 2018.
      vascular
      Only in certain countries. Adapted from Chang 2018.
      LVO/EBDMyocardial perfusion,
      Only in certain countries. Adapted from Chang 2018.
      liver,
      Only in certain countries. Adapted from Chang 2018.
      breast
      Only in certain countries. Adapted from Chang 2018.
      Countries availableNorth America, Europe, Brazil, AsiaNorth America, Europe, Australia, AsiaNorth America, EuropeJapan, South Korea, Norway, Taiwan, China
      Shell/gasPhospholipid/sulphur hexafluoridePhospholipid/perflutrenAlbumin/perflutrenPhospholipid/ perfluorobutane
      ContraindicationsHypersensitivity to sulphur hexafluoride or any inactive ingredientHypersensitivity to perflutrenHypersensitivity to perflutren, blood, blood products or albuminEgg allergy
      Vial supplied10 mL, vial containing 25 mg powder

      5 mL saline
      2 mL vials3 mL vials16 μl of perflubutane microbubbles in one vial

      2 mL sterile water for injection
      StorageRoom temperature2–8°C2–8°CBelow 25°C
      Typical doses2 mL of reconstituted agentWeight based bolus (10 µL/kg) or infusion rate of 4.0 mL/min of 1.3 + 50 mL saline0.5 mLWeight based 15 µL/kg or 0.12 µL MB/kg
      Administration recommendationBolus

      Bolus dose can be repeated once

      Doses varies depending on age and indication
      Bolus or infusion

      Dose varies based on organ being imaged
      Rate not exceed 1 mL/s

      Max dose in 10 min: 5 mL

      Max dose for one study: 8.7 mL
      Bolus or infusion
      MIMI ≤ 0.8MI ≤ 0.8Safety of MI >0.8 has not been testedMI ≤0.8
      AdvantagesApproved for use in pediatric populations

      Reconstitution by hand mixing
      Bolus or infusionResuspend by hand mixingBolus or infusion
      NotesBolus and infusion dosing are used, although package insert only describes bolus dosingRequires 45-s activation on VialMix. Hand agitation thereafter if being used >5 min after activationBolus and infusion dosing are usedRe-injection can be used for Kupffer phase imaging
      EBD = endocardial border definition; LVO = left ventricular opacification; MB = microbubble; MI = mechanical index.
      low asterisk Only in certain countries.Adapted from
      • Chang E.H.
      An Introduction to Contrast-Enhanced Ultrasound for Nephrologists.
      .
      Table 4Size distribution characteristics from the commercially available agents.
      ProductsDN (µm)DV50 (µm)Conc.T. (× 108 MB/mL)MB >10 µm (× 108 MB/mL)MVC (µL/mL)
      SonoVue1.92 ± 0.098.01 ± 0.853.4 ± 0.50.022 ± 0.0066.5 ± 1.2
      Definity1.22 ± 0.038.19 ± 0.7784.0 ± 11.10.143 ± 0.04244.0 ± 9.5
      Optison3.08 ± 0.047.11 ± 0.247.3 ± 0.20.078 ± 0.01735.2 ± 3.0
      Sonazoid2.1 ± 0.12.6 ± 0.112 ± 0.1-8.0 ± 0.6
      DN indicates diameter in number; DV50, diameter in volume; Conc.T., total concentration; MB, microbubbles; MVC, microbubble volume concentration. Adapted from
      • Hyvelin J.M.
      • Gaud E.
      • Costa M.
      • Helbert A.
      • Bussat P.
      • Bettinger T.
      • Frinking P.
      Characteristics and echogenicity of clinical ultrasound contrast agents: An in vitro and in vivo comparison study.
      .

      Optison

      Optison (perflutren protein-type A microspheres injectable suspension) (USP, GE Healthcare, AS, Oslo, Norway) is a sterile non-pyrogenic suspension consisting of microspheres with a perflutren (Octafluoropropane) gas core encapsulated by a 15 nm thick human serum albumin shell (). The reconstitution of Optison is by gentle hand mixing, and the concentration of the reconstituted suspension is 5.0–8.0 × 108 microspheres/mL with an average diameter of 3.0–4.5 µm, and 95% of microspheres are smaller than 10 µm.
      The acoustical characteristics of Optison were investigated previously, and both mathematical modelling and experimental measurements were utilized (
      • Church C.C.
      The effects of an elastic solid surface layer on the radial pulsations of gas bubbles.
      ;
      • Podell S.
      • Burrascano C.
      • Gaal M.
      • Golec B.
      • Maniquis J.
      • Mehlhaff P.
      Physical and biochemical stability of Optison, an injectable ultrasound contrast agent.
      ;
      • Bing C.
      • Hong Y.
      • Hernandez C.
      • Rich M.
      • Cheng B.
      • Munaweera I.
      • Szczepanski D.
      • Xi Y.
      • Bolding M.
      • Exner A.
      • Chopra R.
      Characterization of different bubble formulations for blood-brain barrier opening using a focused ultrasound system with acoustic feedback control.
      ). The albumin shell rigidity of 88.8 MPa and a shell shear viscosity of 0.177 N s/m2 were derived, by assuming the shell as a continuous surface layer of incompressible solid elastic material and by accounting for damping provided by the shell viscosity. Based on data for Albunex (
      • de Jong N.
      • Hoff L.
      Ultrasound scattering properties of Albunex microspheres.
      ), the air-based predecessor of Optison, values for shell elasticity and dilational viscosity can be estimated as 4 N/m and 3.2 × 10−8 kg/s, respectively (
      • Morgan K.E.
      • Allen J.S.
      • Dayton P.A.
      • Chomas J.E.
      • Klibanov A.L.
      • Ferrara K.W.
      Experimental and theoretical evaluation of microbubble behavior: Effect of transmitted phase and bubble size.
      ;
      • Forbes M.M.
      • O'Brien W.D.
      Development of a theoretical model describing sonoporation activity of cells exposed to ultrasound in the presence of contrast agents.
      ). In vitro measurements indicated that the destruction (or gas dissolution) pressure threshold for rapid destruction of Optison was 0.47 MPa (peak-negative pressure) when exposed to 3.5-MHz ultrasound (
      • Podell S.
      • Burrascano C.
      • Gaal M.
      • Golec B.
      • Maniquis J.
      • Mehlhaff P.
      Physical and biochemical stability of Optison, an injectable ultrasound contrast agent.
      ), while a lower threshold was found to contribute to accelerated dissolution (
      • Porter T.M.
      • Smith D.A.B.
      • Holland C.K.
      Acoustic techniques for assessing the Optison destruction threshold.
      ). The flexible and robust characteristics of the albumin shell make Optison an effective ultrasound scatterer, while it can be resilient to destruction in clinical use.
      Optison was launched as the first of the second-generation of UCA in the North American market in January 1998 and in Europe in May of the same year (
      • Jackson A.
      • Castle J.W.
      • Smith A.
      • Kalli C.K.
      Optison albumin microspheres in ultrasound assisted gene therapy and drug delivery.
      ). It is indicated for use in patients with suboptimal echocardiograms to opacify the LV and to improve the delineation of left ventricular endocardial borders (Fig. 1). Off-label use of Optison was reported, including stress echocardiography of patients with suboptimal baseline echocardiograms (
      • Dolan M.S.
      • Riad K.
      • El-Shafei A.
      • Puri S.
      • Tamirisa K.
      • Bierig M.
      • St Vrain J.
      • McKinney L.
      • Havens E.
      • Habermehl K.
      • Pyatt L.
      • Kern M.
      • Labovitz A.J.
      Effect of intravenous contrast for left ventricular opacification and border definition on sensitivity and specificity of dobutamine stress echocardiography compared with coronary angiography in technically difficult patients.
      ,
      • Dolan M.S.
      • Gala S.S.
      • Dodla S.
      • Abdelmoneim S.S.
      • Xie F.
      • Cloutier D.
      • Bierig M.
      • Mulvagh S.L.
      • Porter T.R.
      • Labovitz A.J.
      Safety and efficacy of commercially available ultrasound contrast agents for rest and stress echo a multicenter experience.
      ;
      • Dawson D.
      • Kaul S.
      • Peters D.
      • Rinkevich D.
      • Schnell G.
      • Belcik J.T.
      • Wei K.
      Prognostic value of Dipyridamole stress myocardial contrast echocardiography: Comparison with single photon emission computed tomography.
      ), as well as its non-cardiac clinical applications in liver imaging (i.e., for monitoring the evolving necrosis during thermoablation of liver tumors [
      • Jung E.M.
      • Clevert D.A.
      • Rupp N.
      Contrast-Enhanced Ultrasound with® Optison in Percutaneous Thermoablation of Liver Tumors.
      ]), and it was reported in pediatric patients, such as for the assessment of solid pediatric tumors (
      • McCarville M.B.
      • Kaste S.C.
      • Hoffer F.A.
      • Khan R.B.
      • Walton R.C.
      • Alpert B.S.
      • Furman W.L.
      • Li C.
      • Xiong X.
      Contrast enhanced sonography of malignant pediatric abdominal and pelvic solid tumors: Preliminary safety and feasibility data.
      ) and detection of vesicoureteral reflux (VUR) in children (
      • Ntoulia A.
      • Back S.J.
      • Poznick L.
      • Morgan T.
      • Kerwood J.
      • Christopher E.J.
      • Bellarh R.D.
      • Reid J.R.
      • Jaramillo D.
      • Canning D.A.
      • Darge K.
      Contrast-enhanced voiding urosonography (ceVUS) with the intravesical administration of the ultrasound contrast agent Optison for vesicoureteral reflux detection in children: A prospective clinical trial.
      ).
      Fig 1
      Fig. 1Representative pre-contrast images of a patient who had minimal endocardium visualized on the four chamber view and complete visualization after Optison contrast enhancement: (a) Diastole before contrast. (b) Systole before contrast. (c) Diastole after contrast. (d) Systole after contrast. From
      • Zhao H.
      • O'Quinn R.
      • Ambrose M.
      • Jagasia D.
      • Ky B.
      • Wald J.
      • Ferrari V.A.
      • Kirkpatrick J.N.
      • Han Y.
      Contrast-enhanced echocardiography has the greatest impact in patients with reduced ejection fractions.
      .
      During clinical imaging, Optison can be administered intravenously as either a bolus (0.3–0.5 mL) with a slow rate (not exceeding 1 mL/s) or continuous infusion (10% dilution, 3–5 mL/min). This should be followed by 5–10 mL saline flushes over 10 s to avoid acoustic shadowing and permit steady-state concentration of microbubbles during image acquisition. The recommended Optison dose is 0.5–3.0 mL per patient, with the duration of the left-ventricular contrast of 2.5–4.5 min. A low MI (MI lower than 0.2) in clinical imaging is recommended to avoid microbubble destruction (
      • Porter T.R.
      • Mulvagh S.L.
      • Abdelmoneim S.S.
      • Becher H.
      • Belcik J.T.
      • Bierig M.
      • Choy J.
      • Gaibazzi N.
      • Gillam L.D.
      • Janardhanan R.
      • Kutty S.
      • Leong-Poi H.
      • Lindner J.R.
      • Main M.L.
      • Mathias Jr, W.
      • Park M.M.
      • Senior R.
      • Villanueva F.
      Clinical applications of ultrasonic enhancing agents in echocardiography: 2018 American Society of Echocardiography guidelines update.
      ).

      Definity

      Definity (USA) or Luminity (EU) (perflutren lipid microspheres) (Lantheus Medical Imaging Inc, North Billerica, MA, USA) injectable suspension is supplied as a single-use 2 mL glass vial containing a clear liquid and octafluoropropane (C3F8) gas in the head space. The liquid contains a solution with the three phospholipids DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), DPPA (1,2-Dipalmitoyl-sn-glycero-3-phosphate, sodium salt) and DPPE-MPEG5000. The product is prepared by emulsification activation using a mechanical shaking device (Vialmix) Lantheus Medical Imaging Inc, North Billerica, MA, USA) for 45 s. After activation, a 1 mL milky-white liquid suspension is obtained containing 84.0 ± 11.1 × 108 microspheres/mL (44.0 µL/mL volume concentration) with the number mean diameter of 1.22 ± 0.03 µm and volume median diameter of 8.19 ± 0.77 µm (
      • Hyvelin J.M.
      • Gaud E.
      • Costa M.
      • Helbert A.
      • Bussat P.
      • Bettinger T.
      • Frinking P.
      Characteristics and echogenicity of clinical ultrasound contrast agents: An in vitro and in vivo comparison study.
      ). The viscoelastic shell properties for Definity microbubbles have been estimated at high (12–29 MHz) and at low (5–15 MHz) ultrasound frequencies (
      • Goertz D.E.
      • de Jong N.
      • van der Steen A.F.W.
      Attenuation and size distribution measurements of Definity and manipulated Definity populations.
      ,
      • Faez T.
      • Goertz D.
      • de Jong N.
      Characterization of Definity ultrasound contrast agent at frequency range of 5-15 MHz.
      ). At the lower frequencies, values corresponding to 0.82 N/m for the shell elasticity and 4.0 × 10−9 kg/s for dilational viscosity have been reported (
      • Faez T.
      • Goertz D.
      • de Jong N.
      Characterization of Definity ultrasound contrast agent at frequency range of 5-15 MHz.
      ), which are significantly lower compared with those mentioned above for Optison.
      Definity is one of the second-generation microbubble contrast agents approved for left ventricular opacification and left ventricular endocardial border detection. It was approved in July 2001 by U.S. Food and Drug Administration (FDA) () and in September 2006 by the European Medicine Agency under the name Luminity ( [EPAR] summary for the public). The pharmacokinetics of an intravenous dose of activated Definity was evaluated in healthy humans and those with chronic obstructive pulmonary disease. The perflutren gas is cleared by the lungs in the expired air in an unchanged state. The rapid elimination of perflutren gas in the expired air was also consistent with the rapid disappearance of ultrasound contrast enhancement after activated Definity administration (
      • Abdelmoneim S.S.
      • Mulvagh S.L.
      Perflutren lipid microsphere injectable suspension for cardiac ultrasound.
      ).
      Beyond enhanced endocardial visualization, Definity is used for quantification of LV volumes and ejection fraction (
      • Mulvagh S.L.
      • Rakowski H.
      • Vannan M.A.
      • Abdelmoneim S.S.
      • Becher H.
      • Bierig S.M.
      • Burns P.N.
      • Castello R.
      • Coon P.D.
      • Hagen M.E.
      • Jollis J.G.
      • Kimball T.R.
      • Kitzman D.W.
      • Kronzon I.
      • Labovitz A.J.
      • Lang R.M.
      • Mathew J.
      • Moir W.S.
      • Nagueh S.F.
      • Pearlman A.S.
      • Perez J.E.
      • Porter T.R.
      • Rosenbloom J.
      • Strachan G.M.
      • Thanigaraj S.
      • Wei K.
      • Woo A.
      • Yu E.H.
      • Zoghbi W.A.
      American Society of Echocardiography consensus statement on the clinical applications of ultrasonic contrast agents in echocardiography.
      ). Moreover, the diagnoses of apical LV pathology (e.g., apical variant of hypertrophic cardiomyopathy and thrombus), post–myocardial infarction complications (e.g., LV rupture or pseudoaneurysm, or ventricular septal defect) and intracardiac masses (
      • Porter T.R.
      • Abdelmoneim S.S.
      • Belcik J.T.
      • McCulloch M.L.
      • Mulvagh S.L.
      • Olson J.J.
      • Porcelli C.
      • Tsutsui J.M.
      • Wei K.
      Guidelines for the cardiac sonographer in the performance of contrast echocardiography: A focused update from the American Society of Echocardiography.
      ) are significantly enhanced (Fig. 2). In addition to its cardiac indications, Definity is also approved for imaging of the liver and kidney in Canada and Australia. Several studies have shown its efficacy in evaluation of liver lesions as a vascular phase agent (
      • Claudon M.
      • Dietrich C.F.
      • Choi B.I.
      • Cosgrove D.O.
      • Kudo M.
      • Nolsoe C.P.
      • Piscaglia F.
      • Wilson S.R.
      • Barr R.G.
      • Chammas M.C.
      • Chaubal N.G.
      • Chen M.H.
      • Clevert D.A.
      • Correas J.M.
      • Ding H.
      • Forsberg F.
      • Fowlkes J.B.
      • Gibson R.N.
      • Goldberg B.B.
      • Lassau N.
      • Leen E.L.
      • Mattrey R.F.
      • Moriyasu F.
      • Solbiati L.
      • Weskott H.P.
      • Xu H.X.
      Guidelines and good clinical practice recommendations for contrast enhanced Ultrasound (CEUS) in the liver -update 2012: A WFUMB-EFSUMB initiative in cooperation with representatives of AFSUMB, AIUM, ASUM, FLAUS and ICUS.
      ).
      Fig 2
      Fig. 2Unenhanced (left) and real-time very low MI Definity contrast-enhanced (right) images of a patient with an apical mass. Real-time very low MI contrast-enhanced imaging delineates perfused and non-perfused areas due to endomyocardial fibrosis with thrombus formation (arrow). From
      • Porter T.R.
      • Abdelmoneim S.S.
      • Belcik J.T.
      • McCulloch M.L.
      • Mulvagh S.L.
      • Olson J.J.
      • Porcelli C.
      • Tsutsui J.M.
      • Wei K.
      Guidelines for the cardiac sonographer in the performance of contrast echocardiography: A focused update from the American Society of Echocardiography.
      . MI = mechanical index.
      As mentioned by
      • Abdelmoneim S.S.
      • Mulvagh S.L.
      Perflutren lipid microsphere injectable suspension for cardiac ultrasound.
      , off-label use of Definity has been reported for the use with stress echocardiography improving the diagnostic accuracy of stress echo in the diagnosis of coronary artery disease (
      • Moir S.
      • Haluska B.A.
      • Jenkins C.
      • Fathi R.
      • Marwick T.H.
      Incremental benefit of myocardial contrast to combined dipyridamole-exercise stress echocardiography for the assessment of coronary artery disease.
      ;
      • Tsutsui J.M.
      • Elhendy A.
      • Xie F.
      • O'Leary E.L.
      • McGrain A.C.
      • Porter T.R.
      Safety of dobutamine stress real-time myocardial contrast echocardiography.
      ;
      • Plana J.C.
      • Mikati I.A.
      • Dokainish H.
      • Lakkis N.
      • Abukhalil J.
      • Davis R.
      • Hetzell B.C.
      • Zoghbi W.A.
      A randomized cross-over study for evaluation of the effect of image optimization with contrast on the diagnostic accuracy of dobutamine echocardiography in coronary artery disease: The OPTIMIZE trial.
      ;
      • Dolan M.S.
      • Gala S.S.
      • Dodla S.
      • Abdelmoneim S.S.
      • Xie F.
      • Cloutier D.
      • Bierig M.
      • Mulvagh S.L.
      • Porter T.R.
      • Labovitz A.J.
      Safety and efficacy of commercially available ultrasound contrast agents for rest and stress echo a multicenter experience.
      ) primarily to identify wall motion abnormalities in clinically indicated stress echo studies. Moreover, a growing number of published articles have documented the use of Definity for the assessment of myocardial perfusion to detect perfusion abnormalities both at rest and in conjunction with exercise and pharmacologic stress echocardiography (
      • Abdelmoneim S.S.
      • Mankad S.V.
      • Bernier M.
      • Dhoble A.
      • Hagen M.E.
      • Ness S.C.
      • Chandrasekaran K.
      • Pellikka P.A.
      • Oh J.K.
      • Mulvagh S.L.
      Microvascular function in Takotsubo cardiomyopathy with contrast echocardiography: Prospective evaluation and review of literature.
      ,
      • Abdelmoneim S.S.
      • Wijdicks E.F.
      • Lee V.H.
      • Daugherty W.P.
      • Bernier M.
      • Oh J.K.
      • Pellikka P.A.
      • Mulvagh S.L.
      Real-time myocardial perfusion contrast echocardiography and regional wall motion abnormalities after aneurysmal subarachnoid hemorrhage. Clinical article.
      ).

      SonoVue

      SonoVue (sulphur hexafluoride [SF6] microbubbles) (Bracco Imaging S.p.A., Colleretto Giacosa, Italy) or Lumason (sulphur hexafluoride lipid-type A microspheres) (Bracco Diagnostics Inc., Monroe Township, NJ, USA), is supplied as a kit containing a single-use septum-sealed vial of phospholipid lyophilized powder and SF6 headspace, a pre-filled syringe with 5 mL sodium chloride 0.9% injection (diluent) and a mini-spike transfer system (
      • Hyvelin J.M.
      • Gaud E.
      • Costa M.
      • Helbert A.
      • Bussat P.
      • Bettinger T.
      • Frinking P.
      Characteristics and echogenicity of clinical ultrasound contrast agents: An in vitro and in vivo comparison study.
      ). The lyophilizate consists of polyethylene glycol 4000 and phospholipids DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt) (
      • Schneider M.
      • Arditi M.
      • Barrau M.B.
      • Brochot J.
      • Broillet A.
      • Ventrone R.
      • Yan F.
      BR1: A new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles.
      ). The product is prepared by reconstitution of the lyophilized cake and by shaking the vial for 20 s to obtain a homogenous milky-white suspension of microbubbles, which can be stored for up to 6 h. After reconstitution, the product contains approximately 3.4 ± 0.5 × 108 microspheres/mL (6.5 µL/mL volume concentration) with the number mean diameter of 1.9 ± 0.1 µm and volume median diameter of 8.0 ± 0.9 µm (
      • Hyvelin J.M.
      • Gaud E.
      • Costa M.
      • Helbert A.
      • Bussat P.
      • Bettinger T.
      • Frinking P.
      Characteristics and echogenicity of clinical ultrasound contrast agents: An in vitro and in vivo comparison study.
      ).
      SonoVue is mostly administered as intravenous bolus injection followed by a 5–10 mL saline flush and should be gently agitated before administration. The clinical dose recommended for a single injection is 34 µL/kg (i.e., 2.4 mL for a 70 kg person) (
      • Hyvelin J.M.
      • Gaud E.
      • Costa M.
      • Helbert A.
      • Bussat P.
      • Bettinger T.
      • Frinking P.
      Characteristics and echogenicity of clinical ultrasound contrast agents: An in vitro and in vivo comparison study.
      ); however, the optimal dose may depend on the individual patient and possibly on the scanner technology used. Repeated injection is possible, but in some situations, it is preferred to extend the examination under steady-state conditions (e.g., to prolong the duration of hepatic parenchymal enhancement [
      • Quaia E.
      • Gennari A.G.
      • Angileri R.
      • Cova M.A.
      Bolus versus continuous infusion of microbubble contrast agent for liver ultrasound by using an automatic power injector in humans: A pilot study.
      ] or for measurement of blood flow parameters for assessment of oncologic response to therapy using the destruction-replenishment technique [
      • Dietrich F.
      • Averkiou M.
      • Bachmann M.
      • Barr R.G.
      • Burns P.N.
      • Calliada F.
      • Cantisani V.
      How to perform contrast-enhanced Ultrasound (CEUS).
      ]). For this, a dedicated infusion pump (Vueject, Bracco Imaging, Milan, Italy) with a rotating syringe holder to avoid decantation of the microbubbles has been developed. In this case, SonoVue can be administered as a continuous infusion at a rate of about 1 mL/min depending on the enhancement level required (
      • Schneider M.
      Bubbles in echocardiography: Climbing the learning curve.
      ,
      • Greis C.
      Technology overview. SonoVue (Bracco, Milan).
      ).
      Pharmacokinetics of SonoVue was assessed in humans showing that SF6 is rapidly removed from the blood by the pulmonary route. After intravenous injection, the microbubbles are submitted to systemic pressure variations during the cardiac cycle. The high molecular weight gas SF6, having a low solubility in water and blood, confers good pressure resistance to SonoVue microbubbles (
      • Schneider M.
      • Arditi M.
      • Barrau M.B.
      • Brochot J.
      • Broillet A.
      • Ventrone R.
      • Yan F.
      BR1: A new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles.
      ,
      • Bokor D.
      Diagnostic efficacy of SonoVue.
      ).
      Acoustic properties such as attenuation, backscatter and non-linear behavior of SonoVue microbubbles have been extensively studied (
      • Schneider M.
      • Arditi M.
      • Barrau M.B.
      • Brochot J.
      • Broillet A.
      • Ventrone R.
      • Yan F.
      BR1: A new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles.
      ;
      • Gorce J.M.
      • Arditi M.
      • Schneider M.
      Influence of bubble size distribution on the echogenicity of ultrasound contrast agents: A study of SonoVue.
      ;
      • VanderMeer S.M.
      • Versluis M.
      • Lohse D.
      • Chin C.T.
      • Bouakaz A.
      • de Jong N.
      The resonance frequency of SonoVue™ as observed by high-speed optical imaging.
      ;
      • Biagi E.
      • Breschi L.
      • Vannacci E.
      • Masotti L.
      Stable and transient subharmonic emissions from isolated contrast agent microbubbles.
      ;
      • Chetty K.
      • Stride E.
      • Sennoga C.A.
      • Hajnal J.V.
      • Eckersley R.J.
      High-speed optical observations and simulation results of SonoVue microbubbles at low-pressure insonation.
      ;
      • Tu J.
      • Guan J.
      • Qiu Y.
      • Matula T.J.
      Estimating the shell parameters of SonoVue microbubbles using light scattering.
      ). Visco-elastic shell parameter values of 0.55 N/m for the shell elasticity and 7.2 × 10−9 kg/s for dilational viscosity have been reported (
      • Gorce J.M.
      • Arditi M.
      • Schneider M.
      Influence of bubble size distribution on the echogenicity of ultrasound contrast agents: A study of SonoVue.
      ,
      • Marmottant P.
      • VanderMeer S.
      • Emmer M.
      • Versluis M.
      • de Jong N.
      • Hilgenfeldtb S.
      • Lohse D.
      A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture.
      ), which are very similar to Definity. Moreover,
      • de Jong N.
      • Emmer M.
      • Chin C.T.
      • Bouakaz A.
      • Mastik F.
      • Lohse D.
      • Versluis M.
      “Compression-only” behavior of phospholipid-coated contrast bubbles.
      reported the extremely non-linear characteristic of compression-only behavior, which was observed during the first high-speed imaging recordings (
      • Chin C.T.
      • Lancée C.
      • Borsboom J.
      • Mastik F.
      • Frijlink M.E.
      • de Jong N.
      • Versluis M.
      • Lohse D.
      Brandaris 128: A digital 25 million frames per second camera with 128 highly sensitive frames.
      ) and appeared to be a typical characteristic of shelled microbubbles. In fact, these observations motivated the development of the Marmottant model (
      • Marmottant P.
      • VanderMeer S.
      • Emmer M.
      • Versluis M.
      • de Jong N.
      • Hilgenfeldtb S.
      • Lohse D.
      A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture.
      ) for phospholipid-shelled microbubble dynamics and enforced the fundamental understanding of the success of low-MI contrast-specific imaging techniques in combination with SonoVue.
      SonoVue has clinically been approved in Europe (2001) and China (2004) for use in adult patients with suboptimal echocardiograms to opacify the left ventricular chamber and to improve the delineation of the left ventricular endocardial border, for focal liver lesion and for focal breast lesion characterization ( for the public). In 2014, Lumason obtained approval in the United States for adult patients with suboptimal echocardiograms to opacify the left ventricular chamber and to improve the delineation of the left ventricular endocardial border; in March 2016 it was the first UCA receiving FDA approval for characterizing focal liver lesions in adult and pediatric patients (). Recently, approval for ultrasonography of the urinary tract for the evaluation of suspected or known VUR in pediatric patients was obtained in the United States (2016), Europe (2017) and China (2018). Aside from approved indications, a wide variety of off-label use has been listed primarily for SonoVue in adult (
      • Sidhu P.S.
      • Cantisani V.
      • Dietrich C.F.
      • Gilja O.H.
      • Saftoiu A.
      • Bartels E.
      • Bertolotto M.
      • Calliada F.
      • Clevert D.A.
      • Cosgrove D.
      • Deganello A.
      • D'Onofrio M.
      • Drudi F.M.
      • Freeman S.
      • Harvey C.
      • Jenssen C.
      • Jung E.M.
      • Klauser A.S.
      • Lassau N.
      • Meloni M.F.
      • Leen E.
      • Nicolau C.
      • Nolsoe C.
      • Piscaglia F.
      • Prada F.
      • Prosch H.
      • Radzina M.
      • Savelli L.
      • Weskott H.P.
      • Wijkstra H.
      The EFSUMB guidelines and recommendations for the clinical practice of contrast-enhanced ultrasound (CEUS) in non-hepatic applications: Update 2017 (long version).
      ) and pediatric patients (
      • Sidhu P.S.
      • Cantisani V.
      • Deganello A.
      • Dietrich C.F.
      • Duran C.
      • Franke D.
      • Harkanyi Z.
      • Kosiak W.
      • Miele V.
      • Ntoulia A.
      • Piskunowicz M.
      • Sellars M.E.
      • Gilja O.H.
      Role of contrast-enhanced ultrasound (CEUS) in paediatric practice: An EFSUMB position statement.
      ). Some of the most popular clinical applications of SonoVue are for the diagnosis of focal liver lesions, guidance during ablative treatment and follow-up of liver tumors (
      • Ferraioli G.
      • Meloni M.F.
      Contrast-enhanced ultrasonography of the liver using SonoVue.
      ) (Fig. 3).
      Fig 3
      Fig. 3Focal nodular hyperplasia (FNH) in a 36-y-old woman without a history of liver disease. (a) Spoke-wheel–like enhancement with centrifugal filling in the early arterial phase 15 s after SonoVue injection. (b) In the delayed phase (3 min after injection), the lesion exhibits sustained enhancement except for a central scar (arrow). From
      • Ferraioli G.
      • Meloni M.F.
      Contrast-enhanced ultrasonography of the liver using SonoVue.
      .

      Sonazoid

      Sonazoid microspheres (GE Healthcare, Buckinghamshire, UK; Daiichi Sankyo, Tokyo, Japan) is a second-generation UCA that has been approved in Japan (2006), South Korea (2012), Norway (2014), Taiwan (2017) and mainland China (2018) for contrast-enhanced sonography of focal liver lesions. In Japan, it was approved for contrast-enhanced imaging of breast lesions in 2012.
      Sonazoid is formulated as a lyophilized powder for injection that consists of perfluorobutane (C4F10) microspheres stabilized by a monomolecular membrane of hydrogenated egg yolk phosphatidyl serine (HEPS). The product is reconstituted before use with sterile water through a supplied vented filter spike (5 µm) followed by manual mixing for 1 min. After reconstitution, the product appears as a milky white, homogeneous suspension, containing approximately 1.2 ± 0.1 × 109 microspheres/mL (8 µL/mL volume concentration) with the number mean diameter of 2.1 ± 0.1 µm and volume median diameter of 2.6 ± 0.1 µm (
      • Sontum P.C.
      Physicochemical characteristics of Sonazoid, a new contrast for ultrasound imaging.
      ). Characteristic acoustic properties of Sonazoid microbubbles were described by
      • Katiyar A.
      • Sarkar K.
      Excitation threshold for subharmonic generation from contrast microbubbles.
      using various models for encapsulated microbubbles. Visco-elastic shell parameter estimations obtained with the Marmottant model (
      • Marmottant P.
      • VanderMeer S.
      • Emmer M.
      • Versluis M.
      • de Jong N.
      • Hilgenfeldtb S.
      • Lohse D.
      A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture.
      ) resulted in values of 0.5–0.6 N/m for the shell elasticity and 1.2 × 10–8–1.6 × 10–8 kg/s for dilational viscosity (
      • Faez T.
      • Goertz D.
      • de Jong N.
      Characterization of Definity ultrasound contrast agent at frequency range of 5-15 MHz.
      ); the dilational viscosity is 2–3 times higher compared to SonoVue and Definity, respectively. Sonazoid is highly resistant to overpressures; pressure stress up to 300 mmHg is well tolerated by both concentrated and diluted suspensions (
      • Sontum P.C.
      Physicochemical characteristics of Sonazoid, a new contrast for ultrasound imaging.
      ). The composition of the suspension, physicochemical properties and visco-elastic properties of the stabilizing membrane showed a low variance from vial to vial and from batch to batch, which ensures the consistency in the biologic behavior and clinical efficacy of Sonazoid (
      • Sontum P.C.
      • Østensen J.
      • Dyrstad K.
      • Hoff L.
      Acoustic properties of NC100100 and their relationship with the microsphere size distribution.
      ;
      • Hoff L.
      • Sontum P.C.
      • Hovem J.M.
      Oscillations of polymeric microbubbles: Effect of the encapsulating shell.
      ;
      • Sontum P.C.
      Physicochemical characteristics of Sonazoid, a new contrast for ultrasound imaging.
      ).
      The pharmacokinetics and clinical assessment of Sonazoid showed a very good tolerance of the recommended clinical dose of 0.12 µL perfluorobutane microbubbles/kg weight in healthy adults (
      • Li P.
      • Hoppmann S.
      • Du P.
      • Li H.
      • Evans P.
      • Moestue S.
      • Yu W.
      • Dong F.
      • Liu H.
      • Liu L.
      Pharmacokinetics of perfluorobutane after intra-venous bolus injection of Sonazoid in healthy Chinese volunteers.
      ). Low MI (around 0.2 depending on the scanners) together with contrast-specific non-linear imaging methods should be used to avoid microbubble destruction. As for any agent, the dosage and scanner settings should be adjusted based on patient's body habitus, tissue attenuation, depth of lesions and specific requirements for repetitive injection.
      With clinical imaging of Sonazoid in liver, two phases of contrast enhancement are observed: a vascular phase, followed by a post-vascular phase or Kupffer phase. During Kupffer phase imaging, or parenchyma-specific imaging, the normal hepatic parenchyma is enhanced, and malignant lesions appear as clear contrast defects (
      • Moriyasu F.
      • Itoh K.
      Efficacy of perflubutane microbubble-enhanced ultrasound in the characterization and detection of focal liver lesions: Phase 3 multicenter clinical trial.
      ). The pattern of vascular phase and Kupffer phase enhancement can be used to better characterize focal liver lesions and to detect or rule out the presence of lesions (
      • Correas J.M.
      • Low G.
      • Needleman L.
      • Robbin M.L.
      • Cosgrove D.
      • Sidhu P.S.
      • Harvey C.J.
      • Albrecht T.
      • Jakobsen J.A.
      • Brabrand K.
      • Jenett M.
      • Bates J.
      • Claudon M.
      • Leen E.
      Contrast enhanced ultrasound in the detection of liver metastases: A prospective multi-center dose testing study using a perfluorobutane microbubble contrast agent (NC100100).
      ;
      • Jo P.
      • Jang H.
      • Burns P.
      • Burak K.
      • Kim T.
      • Wilson S.
      Integration of contrast-enhanced US into a multimodality approach to imaging of nodules in a cirrhotic liver: How to do it.
      ;
      • Zhai H.Y.
      • Liang P.
      • Yu J.
      • Cao F.
      • Kuang M.
      • Liu F.Y.
      • Liu F.Y.
      • Zhu X.Y.
      Comparison of Sonazoid and Sonovue in the diagnosis of focal liver lesions: A preliminary study.
      ). Additionally, the prolonged time window during Kupffer phase imaging enables repeated scanning up to 10–60 min after injection, improving lesion detection.
      • Kudo M.
      Defect reperfusion imaging with Sonazoid: A breakthrough in hepatocellular carcinoma.
      developed a defect reperfusion imaging method during which the contrast is re-injected based on enhancement defects detected during the stable Kupffer phase, providing additional information on arterial enhancement to improve the accurate diagnostic and treatment strategy of hepatocellular carcinoma. Figure 4 shows exemplary images after Sonozoid injection of liver metastasis and focal nodular hyperplasia (FNH). Off-label use of Sonazoid has been reported such as for the detection of sentinel lymph nodes (
      • Shimazu K.
      • Ito T.
      • Uji K.
      • Miyake T.
      • Aono T.
      • Motomura K.
      • Naoi Y.
      • Shimomura A.
      • Shimoda M.
      • Kagara N.
      • Kim S.J.
      • Noguchi S.
      Identification of sentinel lymph nodes by contrast-enhanced ultrasonography with Sonazoid in patients with breast cancer: A feasibility study in three hospitals.
      ).
      Fig 4
      Fig. 4Sonazoid-enhanced imaging of focal liver lesions. (a–d) A 75-y-old man with liver metastasis; (e–h) A 38-y-old woman with FNH. (a) A CE-CT image displays slight peripheral enhancement with hypoattenuating centre. (b) Pre-contrast harmonic B-mode shows isoechoic metastasis. (c) Arterial phase post–Sonazoid injection shows metastasis with non-enhancing regions. (d) Kupffer phase at 10 min post–Sonazoid injection shows metastasis non-enhancing with well-defined rim. (e) FNH hyperintense representation in the venous/delayed phase on CE-CT. (f) Pre-contrast harmonic B-mode image of FNH. (g) Arterial phase shows well-defined lesion margins and central scarring. (h) Kupffer phase at 10 min post–Sonazoid injection shows homogeneous iso-enhancement of FNH. From: An Efficacy and Safety Study of Sonazoid and SonoVue in Participants With Focal Liver Lesions, Undergoing Pre- and Post-Contrast Ultrasound Imaging (ClinicalTrials.gov Identifier: NCT03335566). This phase 3 prospective study was conducted at 17 centres in greater China and Korea (May 2014 to April 2015). The study was approved by an independent ethics committee or independent review board at each clinical site according to national or local regulations. Written informed consent was obtained from all study participants. CE-CT = contrast-enhanced computed tomography; FNH = focal nodular hyperplasia.
      All commercial contrast agents meet the criteria as defined more than 3 decades ago for successful clinical utility after intravenous injections, particularly in terms of demonstrating an excellent safety profile (
      • Main M.L.
      • Goldman J.H.
      • Rayburn P.A.
      Thinking outside the “box”-The ultrasound contrast controversy.
      ; Claudon et al. 2012). Variations in composition, physico-chemical and shell visco-elastic properties between the agents, as reported above, allows for specific applications depending on the agent. For example, the extremely non-linear behavior of SonoVue and Definity at low MI (lower than 0.1) is exploited (e.g., for real-time imaging in deep tissue where the ultrasound beam can be severely affected by attenuation). On the other hand, Sonazoid's high persistence and resistance to acoustic pressure allows its microbubbles to facilitate a specific Kupffer uptake (Fig. 5) not seen with the other marketed agents (
      • Watanabe R.
      • Matsumura M.
      • Munemasa T.
      • Fujimaki M.
      • Suematsu M.
      Mechanism of hepatic parenchyma-specific contrast of microbubble-based contrast agent for ultrasonography: Microscopic studies in rat liver.
      ;
      • Yanagisawa K.
      • Moriyasu F.
      • Miyahara T.
      • Yuki M.
      • Iijima H.
      Phagocytosis of ultrasound contrast agent microbubbles by Kupffer cells.
      ).
      Fig 5
      Fig. 5Confocal laser scanning microscopy (CLSM) and phase-contrast microscopy indicating uptake/phagocytic process of Sonazoid microbubbles by Kupffer cells. (a) Microbubbles detected as reflected light (green spots). (b) Stained cytoplasm of Kupffer cells (red). (c) Differential interference contrast image. (d) An overlay of panels a, b and c. Strong reflected light spots from Sonazoid microbubbles were observed in the cytoplasm of isolated Kupffer cells. Single microbubble attached to the outside of cell membrane could be discriminated, as it was not surrounded by the cytoplasm (arrowhead). From
      • Watanabe R.
      • Matsumura M.
      • Munemasa T.
      • Fujimaki M.
      • Suematsu M.
      Mechanism of hepatic parenchyma-specific contrast of microbubble-based contrast agent for ultrasonography: Microscopic studies in rat liver.
      .

      Future improvement

      While contrast-specific detection technology has seen dramatic progress since the introduction of UCA, successful clinical translation of new developments has been limited during the same period, while understanding of microbubble physical, chemical and biologic behavior has improved substantially. Indeed, technology has made enormous advancements with the introduction of, for example, targeted microbubbles for molecular imaging and for therapeutic applications, but none of these developments have resulted in new clinical agents specific to these applications. This section will focus on a new advancement that can be employed to optimize microbubble performance.

      Monodisperse microbubbles

      The production methods of commercially available contrast agents result in microbubble suspensions with inherently polydisperse size distributions, typically ranging from 1–10 µm in diameter (
      • Stride E.
      • Saffari N.
      Microbubble ultrasound contrast agents: A review.
      ). Microbubbles excited near their resonance frequency exhibit the strongest relative radial excursion and thereby produce the strongest non-linear echo response (
      • Leighton T.G.
      The acoustic bubble.
      ;
      • Segers T.
      • de Jong N.
      • Versluis M.
      Uniform scattering and attenuation of acoustically sorted ultrasound contrast agents: Modeling and experiments.
      ). Clinical ultrasound scanners operate at a narrow frequency bandwidth relative to the resonance frequencies of the microbubbles present in a typical polydisperse UCA. Consequently, only a small fraction of the microbubbles is excited near their resonance frequency and contributes to the generation of the non-linear echo. Indeed, it has frequently been suggested that the sensitivity of contrast-enhanced ultrasound imaging can be improved by narrowing down the microbubble size distribution (
      • Talu E.
      • Hettiarachchi K.
      • Zhao S.
      • Powell R.L.
      • Lee A.P.
      • Longo M.L.
      • Dayton P.A.
      Tailoring the size distribution of ultrasound contrast agents: Possible method for improving sensitivity in molecular imaging.
      ;
      • Streeter J.E.
      • Gessner R.
      • Miles I.
      • Dayton P.A.
      Improving sensitivity in ultrasound molecular imaging by tailoring contrast agent size distribution: in vivo studies.
      ). In fact, a sensitivity increase of 2–3 orders of magnitude has been measured in vitro for resonantly driven monodisperse microbubbles compared with a polydisperse agent (
      • Segers T.
      • Kruizinga P.
      • Kok M.
      • Lajoinie G.
      • de Jong N.
      • Versluis M.
      Monodisperse versus polydisperse ultrasound contrast agents: Non-linear response, sensitivity, and deep tissue imaging potential.
      ).

      Monodisperse microbubbles for molecular imaging

      The increased sensitivity provided by monodisperse microbubbles may particularly be of interest in ultrasound molecular imaging applications using ligand-bearing microbubbles, which can be targeted to specific receptors expressed on endothelial cells (
      • Klibanov A.L.
      Microbubble contrast agents: Targeted ultrasound imaging and ultrasound-assisted drug-delivery applications.
      ). Successful binding of these molecular agents is non-trivial and depends on, for example, ligand-receptor affinity, ligand density, level of receptor expression, vessel diameter and wall shear rate (
      • Tranquart F.
      • Arditi M.
      • Bettinger T.
      • Frinking P.
      • Hyvelin J.M.
      • Nunn A.
      • Pochon S.
      • Tardy I.
      Ultrasound contrast agents for ultrasound molecular imaging.
      ). Although promising pre-clinical results have been reported demonstrating high ligand-receptor affinity and specificity (
      • Pochon S.
      • Tardy I.
      • Bussat P.
      • Bettinger T.
      • Brochot J.
      • von Wronski M.
      • Passantino L.
      • Schneider M.
      BR55: A lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.
      ;
      • Tardy I.
      • Pochon S.
      • Theraulaz M.
      • Emmel P.
      • Passantino L.
      • Tranquart F.
      • Schneider M.
      Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55.
      ), clinical translation has not yet resulted in approval of any ultrasound molecular imaging agent, despite positive initial clinical results (
      • Smeenge M.
      • Tranquart F.
      • Mannaerts C.K.
      • de Reijke T.M.
      • van de Vijver M.J.
      • Laguna M.P.
      • Pochon S.
      • de la Rosette J.J.M.C.H.
      • Wijkstra H.
      First-in-human ultrasound molecular imaging with a VEGFR2-specific ultrasound molecular contrast agent (BR55) in prostate cancer: A safety and feasibility pilot study.
      ;
      • Willmann J.K.
      • Bonomo L.
      • Testa A.C.
      • Rinaldi P.
      • Rindi G.
      • Valluru K.S.
      • Petrone G.
      • Martini M.
      • Lutz A.M.
      • Gambhir S.S.
      Ultrasound molecular imaging with BR55 in patients with breast and ovarian lesions: First-in-human results.
      ). It has been suggested that this is partially due to the polydisperse character of these agents, resulting in a suboptimal performance even at an agent dose corresponding to approximately 10 times the typical imaging dose used with a non-targeted agent such as SonoVue (
      • Frinking P.
      • Tardy I.
      • Théraulaz M.
      • Arditi M.
      • Powers J.
      • Pochon S.
      • Tranquart F.
      Effects of acoustic radiation force on the binding efficiency of BR55, a VEGFR2-specific ultrasound contrast agent.
      ). The suboptimal performance results from the fact that only small fractions of the total injected number of primarily off-resonant microbubbles bind to the target site. Thus, an acoustically uniform and narrowband response of monodisperse microbubbles (
      • Segers T.
      • de Rond L.
      • de Jong N.
      • Borden M.
      • Versluis M.
      Stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at high production rates.
      ) may dramatically improve imaging sensitivity of individually bound microbubbles. Moreover, monodisperse bubbles may allow for successful discrimination of targeted microbubbles from freely circulating ones through spectral changes owing to a resonance frequency shift of the microbubbles of a given size after binding (
      • Overvelde M.
      • Garbin V.
      • Dollet B.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Dynamics of coated microbubbles adherent to a wall.
      ).

      Monodisperse microbubbles for theragnostic applications

      The narrowband and acoustically uniform response of a monodisperse microbubble suspension is furthermore of great interest for emerging theragnostic applications using microbubbles and ultrasound. The echogenic bubble can be tracked in the body while at increasing acoustic pressures it can locally deliver a drug payload (
      • Tsutsui J.M.
      • Xie F.
      • Porter R.T.
      The use of microbubbles to target drug delivery.
      ;
      • Hernot S.
      • Klibanov A.L.
      Microbubbles in ultrasound-triggered drug and gene delivery.
      ;
      • Deelman L.E.
      • Decleves A.E.
      • Rychak J.J.
      • Sharma K.
      Targeted renal therapies through microbubbles and ultrasound.
      ;
      • Carson A.R.
      • McTiernan C.F.
      • Lavery L.
      • Grata M.
      • Leng X.
      • Wang J.
      • Chen X.
      • Villanueva F.S.
      Ultrasound-targeted microbubble destruction to deliver siRNA cancer therapy.
      ;
      • Dewitte H.
      • Vanderperren K.
      • Haers H.
      • Stock E.
      • Duchateau L.
      • Hesta M.
      • Saunders J.H.
      • De Smedt S.C.
      • Lentacker I.
      Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: implications for drug delivery.
      ), induce cell poration (
      • Helfield B.
      • Chen X.
      • Watkins S.C.
      • Villanueva F.S.
      Biophysical insight into mechanisms of sonoporation.
      ), temporarily open the blood–brain barrier (
      • Hynynen K.
      • McDannold N.
      • Vykhodtseva N.
      • Raymond S.
      • Weissleder R.
      • Jolesz F.A.
      • Sheikov N.
      Focal disruption of the blood-brain barrier due to 260-kHz ultrasound bursts: A method for molecular imaging and targeted drug delivery.
      ;
      • Choi J.J.
      • Feshitan J.A.
      • Baseri B.
      • Wang S.
      • Tung Y.S.
      • Borden M.A.
      • Konofagou E.E.
      Microbubble-size dependence of focused ultrasound-induced blood brain barrier opening in mice in vivo.
      ;
      • Konofagou E.E.
      • Tung Y.S.
      • Choi J.
      • Deffieux T.
      • Baseri B.
      • Vlachos F.
      Ultrasound-induced blood-brain barrier opening.
      ) or lyse a blood clot (
      • Molina C.A.
      • Barreto A.D.
      • Tsivgoulis G.
      • Sierzenski P.
      • Malkoff M.D.
      • Rubiera M.
      • Gonzales N.
      • Mikulik R.
      • Pate G.
      • Ostrem J.
      • Singleton W.
      • Manvelian G.
      • Unger E.C.
      • Grotta J.C.
      • Schellinger P.D.
      • Alexandrov A.V.
      Transcranial ultrasound in clinical sonothrombolysis (TUCSON) trial.
      ). Key to all these emerging applications is a precise control over the interaction of microbubbles with the ultrasound wave (
      • Stride E.
      • Edirisinghe M.
      Novel preparation techniques for controlling microbubble uniformity: A comparison.
      ) to induce a maximal therapeutic effect while minimizing possible side effects such as haemorrhage and cell death (
      • Karshafian R.
      • Bevan P.D.
      • Williams R.
      • Samac S.
      • Burns P.N.
      Sonoporation by ultrasound-activated microbubble contrast agents: Effect of acoustic exposure parameters on cell membrane permeability and cell viability.
      ;
      • 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.
      ). Such an accurate control over volumetric microbubble oscillations can be achieved by using monodisperse microbubbles. Furthermore, because of their uniform acoustic response, monodisperse microbubble populations may potentially solve remaining fundamental questions as to the optimal acoustic parameters and corresponding volumetric oscillation amplitudes required to induce therapeutic effects such as endocytosis, sonoporation and cell death (
      • Karshafian R.
      • Bevan P.D.
      • Williams R.
      • Samac S.
      • Burns P.N.
      Sonoporation by ultrasound-activated microbubble contrast agents: Effect of acoustic exposure parameters on cell membrane permeability and cell viability.
      ;
      • 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.
      :
      • 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.
      ).

      Production techniques for monodisperse microbubbles

      For all these reasons, efforts have been made to develop next-generation microbubble agents with a narrow and controllable size distribution. To date, two strategies exist for achieving this. The first approach is based on isolating a subset of sizes from the native size distribution of a polydisperse microbubble suspension. This can be done by mechanical filtration (
      • Emmer M.
      • Vos H.J.
      • Goertz D.E.
      • van Wamel A.
      • Versluis M.
      • de Jong N.
      Pressure-dependent attenuation and scattering of phospholipid-coated microbubbles at low acoustic pressures.
      ) (Fig. 6a), by decantation (
      • Goertz D.E.
      • de Jong N.
      • van der Steen A.F.W.
      Attenuation and size distribution measurements of Definity and manipulated Definity populations.
      ) (Fig. 6b), by centrifugation (
      • Feshitan J.A.
      • Chen C.C.
      • Kwan J.J.
      • Borden M.A.
      Microbubble size isolation by differential centrifugation.
      ) (Fig. 6c) or by pinched flow fractionation (
      • Kok M.P.
      • Segers T.
      • Versluis M.
      Bubble sorting in pinched microchannels for ultrasound contrast agent enrichment.
      ) (Fig. 6d). However, size uniformity obtained by these methods does not necessarily result in acoustic uniformity. However, polydisperse agents can also be sorted to their acoustic resonance in an acoustic bubble sorting chip (
      • Segers T.
      • Versluis M.
      Acoustic bubble sorting for ultrasound contrast agent enrichment.
      ) (Fig. 6e). It has been shown that an acoustically sorted bubble suspension has a uniform acoustic response (
      • Segers T.
      • de Jong N.
      • Versluis M.
      Uniform scattering and attenuation of acoustically sorted ultrasound contrast agents: Modeling and experiments.
      ). A further limitation of the aforementioned sorting methods is that typical native size distributions of polydisperse agents primarily consist of small bubbles (smaller than 2–3 µm in diameter), whereas it has been suggested that larger bubbles (approximately 4–5 µm in diameter) are preferable for therapeutic applications such as blood–brain barrier opening, since these bubbles are resonant to ultrasound frequencies typically used for therapy (lower than 1 MHz) (
      • Choi J.J.
      • Feshitan J.A.
      • Baseri B.
      • Wang S.
      • Tung Y.S.
      • Borden M.A.
      • Konofagou E.E.
      Microbubble-size dependence of focused ultrasound-induced blood brain barrier opening in mice in vivo.
      ).
      Fig 6
      Fig. 6Different strategies to produce microbubbles with a narrow size distribution. (a) Pore filters can be used to filter bubbles to size (
      • Emmer M.
      • Vos H.J.
      • Goertz D.E.
      • van Wamel A.
      • Versluis M.
      • de Jong N.
      Pressure-dependent attenuation and scattering of phospholipid-coated microbubbles at low acoustic pressures.
      ). Bubbles can be sorted using the volume-dependent gravitational force, either directly by (b) decantation or through (c) centrifugation. (d) Pinched flow fractionation is a microfluidic sorting technique. The microbubbles are pinned to the top wall of the pinched segment by a co-flow. Size-selective sorting is achieved through expansion of the pinched segment into the broadened segment. (e) Acoustic bubble sorting sorts bubbles to their acoustic resonance behavior rather than to their size using the primary acoustic radiation induced by a traveling acoustic wave. (f) Microbubbles can be directly synthesized in a flow-focusing device. Figure is based on
      • Segers T.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      In Microbubbles for medical applications.
      ,
      • Segers T.
      • Gaud E.
      • Versluis M.
      • Frinking P.
      High-precision acoustic measurements of the nonlinear dilatational elasticity of phospholipid coated monodisperse microbubbles.
      . a was taken from Aquamarijn, The Netherlands (from https://www.aquamarijn.nl/technology/ with permission).
      The second approach for producing monodisperse microbubbles is through direct bubble formation in a microfluidic flow-focusing device (
      • Ganan-Calvo A.M.
      • Gordillo J.M.
      Perfectly monodisperse microbubbling by capillary ow focusing.
      ;
      • Anna S.L.
      • Bontoux N.
      • Stone H.A.
      Formation of dispersions using “flow focusing” in microchannels.
      ;
      • Garstecki P.
      • Gitlin I.
      • DiLuzio W.
      • Whitesides G.M.
      Formation of monodisperse bubbles in a microfluidic flow-focusing device.
      ;
      • Garstecki P.
      • Stone H.A.
      • Whitesides G.M.
      Mechanism for flow-rate controlled breakup in confined geometries: A route to monodisperse emulsions.
      ;
      • Dollet B.
      • van Hoeve W.
      • Raven J.P.
      • Marmottant P.
      • Versluis M.
      Role of the channel geometry on the bubble pinch-off in flow-focusing devices.
      ). In such a device, a gas thread is focused between two liquid flows through a constriction where it destabilizes because of capillary instability and pinches off to release monodisperse bubbles (Fig. 6f). The bubble size, stability and the generation frequency can now be accurately controlled through the gas pressure, the liquid flow rate and the lipid mixture (
      • Segers T.
      • de Rond L.
      • de Jong N.
      • Borden M.
      • Versluis M.
      Stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at high production rates.
      ,
      • Segers T.
      • Lohse D.
      • Versluis M.
      • Frinking P.
      Universal equations for the coalescence probability and long-term size stability of phospholipid-coated monodisperse microbubbles formed by ow-focusing.
      ,
      • Segers T.
      • Lassus A.
      • Bussat P.
      • Gaud E.
      • Frinking P.
      Improved coalescence stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at elevated temperatures.
      ). Bubble formation rates exceeding 1 million bubbles per second can be achieved using a single nozzle (
      • Castro-Hernandez E.
      • van Hoeve W.
      • Lohse D.
      • Gordillo J.
      Microbubble generation in a co-ow device operated in a new regime.
      ;
      • Segers T.
      • de Rond L.
      • de Jong N.
      • Borden M.
      • Versluis M.
      Stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at high production rates.
      ), and 2.4 mL of a monodisperse microbubble suspension can be produced in 15 min using a microfluidic device as shown in Figure 6f. This would translate into producing a clinically relevant imaging dose in less than 1 min, considering the 2–3 orders of magnitude gain in sensitivity for monodisperse microbubbles compared with SonoVue (
      • Segers T.
      • Kruizinga P.
      • Kok M.
      • Lajoinie G.
      • de Jong N.
      • Versluis M.
      Monodisperse versus polydisperse ultrasound contrast agents: Non-linear response, sensitivity, and deep tissue imaging potential.
      ,
      • Segers T.
      • Lassus A.
      • Bussat P.
      • Gaud E.
      • Frinking P.
      Improved coalescence stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at elevated temperatures.
      ).
      The acoustic response of monodisperse microbubble suspensions was shown to have a narrowband and uniform response (
      • Segers T.
      • de Jong N.
      • Versluis M.
      Uniform scattering and attenuation of acoustically sorted ultrasound contrast agents: Modeling and experiments.
      ,
      • Segers T.
      • de Rond L.
      • de Jong N.
      • Borden M.
      • Versluis M.
      Stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at high production rates.
      , 2018). However, to date, questions remain as to the exact clinical requirements regarding monodispersity in terms of size and acoustic resonance behavior for imaging and especially for therapeutic applications. It is, for example, being debated that the improved sensitivity observed with monodisperse microbubbles may also be obtained with a polydisperse agent by injecting a higher dose (
      • Talu E.
      • Hettiarachchi K.
      • Zhao S.
      • Powell R.L.
      • Lee A.P.
      • Longo M.L.
      • Dayton P.A.
      Tailoring the size distribution of ultrasound contrast agents: Possible method for improving sensitivity in molecular imaging.
      ;
      • Kaya M.
      • Feingold S.
      • Hettiarachchi K.
      • Lee A.P.
      • Dayton P.A.
      Acoustic responses of monodisperse lipid-encapsulated microbubble contrast agents produced by flow focusing.
      ). Future studies are thus required to investigate the full clinical potential and in vivo performance of monodisperse microbubbles. Nevertheless, the current advances look promising, and these can drive forward emerging applications of lipid-coated microbubbles in combination with ultrasound that are currently suboptimal owing to the use of polydisperse agents.

      Conclusion

      Contrast-enhanced ultrasound imaging has seen tremendous progress during the last 3 decades. Historically, the clinical focus was primarily on echocardiography, for which LV opacification (i.e., improving LV endocardial border delineation) was the major indication, while myocardial perfusion was described as the holy grail and considered as a major market for contrast echo. The commercial agents have proven to be successful; however, there is still no approval for myocardial perfusion, and major clinical applications of contrast-enhanced ultrasound are outside cardiology with many relevant off-label opportunities still to be approved. Advancements in understanding microbubble physics, microbubble biophysics and the technological developments in ultrasound equipment with sensitive contrast-specific imaging methodologies have been of crucial importance for broad clinical acceptance of contrast-enhanced ultrasound imaging, turning it into a mature and successful competitor of traditional diagnostic modalities, such as contrast-enhanced MRI and CT. It is expected that for successful development of future opportunities, such as ultrasound molecular imaging and therapeutic applications using microbubbles, new creative developments in microbubble engineering and production dedicated to further optimizing microbubble performance are required, and that they cannot rely on bubble technology developed more 3 three decades ago.

      Conflict of interest disclosure

      Peter Frinking is employee of Tide Microfluidics; Ying Luan and Francois Tranquart are employees of General Elextric Healthcare.

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