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Microbubble Agents: New Directions

      Abstract

      Microbubble ultrasound contrast agents have now been in use for several decades and their safety and efficacy in a wide range of diagnostic applications have been well established. Recent progress in imaging technology is facilitating exciting developments in techniques such as molecular, 3-D and super resolution imaging and new agents are now being developed to meet their specific requirements. In parallel, there have been significant advances in the therapeutic applications of microbubbles, with recent clinical trials demonstrating drug delivery across the blood–brain barrier and into solid tumours. New agents are similarly being tailored toward these applications, including nanoscale microbubble precursors offering superior circulation times and tissue penetration. The development of novel agents does, however, present several challenges, particularly regarding the regulatory framework. This article reviews the developments in agents for diagnostic, therapeutic and “theranostic” applications; novel manufacturing techniques; and the opportunities and challenges for their commercial and clinical translation.

      Key Words

      Background

      Discovery and development of ultrasound contrast agents

      The first published report of an ultrasound contrast agent was by
      • Gramiak R.
      • Shah P.M.
      Echocardiography of the aortic root.
      . They described observations, made by themselves and Dr. Claude Joyner, of enhanced echogenicity of the aortic root after rapid injection of saline (
      • Gramiak R.
      • Shah P.M.
      Echocardiography of the aortic root.
      ). Their hypothesis was that this effect was attributable to the presence of microbubbles, either produced during injection or pre-existing in the liquid. Subsequent studies by
      • Kremkau F.W.
      • Gramiak R.
      • Carstensen E.L.
      • Shah P.M.
      • Kramer D.H.
      Ultrasonic detection of cavitation at catheter tips.
      supported this proposal by demonstrating that no contrast enhancement was observed when the ambient pressure was increased to suppress bubble formation. Similar observations were also reported with other liquids, including indocynanine green (
      • Feigenbaum H.
      • Stone J.M.
      • Lee D.A.
      • Nasser W.K.
      • Chang S.
      Identification of ultrasound echoes from the left ventricle by use of intracardiac injections of indocyanine green.
      ), ether and even whole blood (
      • Ziskin M.C.
      • Bonakdarpour A.
      • Weinstein D.P.
      • Lynch P.R.
      Contrast agents for diagnostic ultrasound.
      ). The duration of the contrast enhancement was, however, extremely short because of the instability of the bubbles. This challenge was addressed by the discovery that a stabilising coating of cross-linked serum albumin could extend the bubble lifetime by inhibiting gas diffusion and reducing surface tension. This in turn gave rise to the first commercial ultrasound contrast agent, Albunex (
      • Feinstein S.B.
      • Ten Cate F.J.
      • Zwehl W.
      • Ong K.
      • Maurer G.
      • Tei C.
      • Shah P.M.
      • Meerbaum S.
      • Corday E.
      Two-dimensional contrast echocardiography: In vitro development and quantitative analysis of echo contrast agents.
      ). The subsequent decades have seen the development of numerous alternative agents, offering improvements in echogenicity and circulation time, and with exciting demonstrations of the potential use of microbubbles in molecular imaging (
      • Klibanov A.L.
      Ultrasound molecular imaging with targeted microbubble contrast agents.
      ) and drug delivery (
      • De Cock I.
      • Zagato E.
      • Braeckmans K.
      • Luan Y.
      • de Jong N.
      • De Smedt S.C.
      • Lentacker I.
      Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis.
      ). Surprisingly, however, only a small number of agents have been successfully translated into clinical use. The aim of this article is to review the state of the art and recent developments in agents for ultrasound imaging, therapy and their combined or “theranostic” applications. In addition, we will discuss the opportunities and challenges for their clinical translation.
      The inclusion criteria for the pre-clinical and clinical studies cited were respectively that IACUC approval was obtained if animals were studied or that informed consent was obtained from each study participant and that each study was approved by an ethics committee or institutional review board. Most studies cited were published in journals for which these criteria were conditions of publication. In studies for which this was not the case, we verified that appropriate statements were included. For a small number of studies we were unable to confirm ethical approval because they were published before the widespread requirement for inclusion of a statement-of-ethics board approval. We, however, have no reason to doubt that the work was compliant with the 1964 Declaration of Helsinki.

      Classes of agents

      The key requirements for an imaging contrast agent are that it should be non-toxic, generate a strong and unique imaging signal and have a sufficient circulation time for diagnostic procedures. For certain applications it is also desirable to be able to target the agent to specific sites and to control whether it remains in the bloodstream or can diffuse into the surrounding tissue. For therapeutic or theranostic applications, the generation of an imaging signal may be of less importance, but there may be the further requirement for attachment of a payload (e.g., drug molecules and/or generation of mechanical, thermal, or chemical effects).

      Microbubbles

      All the contrast agents currently available clinically consist of suspensions of gas microbubbles, having diameters in the range 1–10 µm (Fig. 1) and vial concentrations of approximately 108 to 109 microbubbles/mL. They are typically administered by intravenous injection of 1–2 mL, giving an average blood concentration of ∼105 to 106 microbubbles/mL, which is sufficient to produce strong contrast enhancement even at low ultrasound intensities (<1 Wcm−2). The original Albunex microbubbles contained air, but the so-called “second generation” of contrast agents all contain a high molecular weight, low solubility gas such as perfluoropropane (C3F8) or sulphur hexafluoride (SF6), which significantly enhances bubble stability and hence prolongs the duration of contrast enhancement. Alternative coatings to serum albumin have also been extensively investigated, including cyanoacrylates, biopolymers such as polylactic acid and polylactic-co-glycolic acid (
      • Straub J.A.
      • Chickering D.E.
      • Church C.C.
      • Shah B.
      • Hanlon T.
      • Bernstein H.
      Porous PLGA microparticles: AI-700, an intravenously administered ultrasound contrast agent for use in echocardiography.
      ), palmitic acid (
      • Goldberg B.B.
      • Liu J.B.
      • Burns P.N.
      • Merton D.A.
      • Forsberg F.
      Galactose-based intravenous sonographic contrast agent: Experimental studies.
      ). Most successfully in use are phospholipids (
      • Unger E.
      • Fritz T.
      • Shen D.K.
      • Lund P.
      • Sahn D.
      • Ramaswami R.
      • Matsunaga T.
      • Yellowhair D.
      • Kulik B.
      Gas filled lipid bilayers as imaging contrast agents.
      ), which offer a favorable compromise between microbubble stability and echogenicity. Microbubbles do, however, have limitations for certain applications. They are too large to extravasate before ultrasound exposure and can thus only be used to image the vasculature. In addition, their circulation half-life is relatively short (<5 min) on account of their size (
      • Schneider M.
      Characteristics of SonoVue.
      ). For many conventional imaging procedures, these limitations do not present significant problems. Indeed, for applications in which only vascular imaging is required and high patient throughput is desirable, they are advantageous. They are, however, restrictive for more complex imaging and therapeutic procedures. Pegylation has been demonstrated to improve circulation times but only by a relatively modest amount (
      • 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.
      ). Microbubbles also can be rapidly destroyed once exposed to ultrasound, which again limits their utility for prolonged imaging or therapeutic delivery.
      Fig 1
      Fig. 1Overview of the various classes of agents for ultrasound imaging and therapy. The white scale bar represents 1 µm. (Image of echogenic liposomes reproduced with permission from
      • Hitchcock K.E.
      • Caudell D.N.
      • Sutton J.T.
      • Klegerman M.E.
      • Vela D.
      • Pyne-Geithman G.J.
      • Abruzzo T.
      • Cyr P.E.P.
      • Geng Y.J.
      • McPherson D.D.
      • Holland C.K.
      Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model.
      .] Copyright 2016, Elsevier. Image of nanobubbles reproduced with permission from
      • Zhang J.
      • Chen Y.
      • Deng C.
      • Zhang L.
      • Sun Z.
      • Wang J.
      • Yang Y.
      • Lv Q.
      • Han W.
      • Xie M.
      The optimized fabrication of a novel nanobubble for tumor imaging.
      . Copyright 2019, Frontiers in Pharmacology. Image of solid nuclei reproduced with permission from
      • Kwan J.J.
      • Myers R.
      • Coviello C.M.
      • Graham S.M.
      • Shah A.R.
      • Stride E.
      • Carlisle R.C.
      • Coussios C.C.
      Ultrasound-propelled nanocups for drug delivery.
      . Copyright 2015, Wiley).

      Phase-change liquid droplets

      One of the early second-generation contrast agents, EchoGen (
      • Grayburn P.
      Perflenapent emulsion (EchoGen): A new long-acting phase-shift agent for contrast echocardiography.
      ) consisted of an emulsion of sub-micrometer perfluorocarbon liquid droplets that could be vaporised to form microbubbles after injection. The liquid-liquid emulsion offered improved storage stability and was believed to enable increased in vivo microbubble stability and hence contrast persistence. EchoGen was not ultimately commercially successful but a similar principle has been adopted in the development of so-called “phase shift emulsions” or “acoustically activated vaporisation agents,” with the aim of addressing the size limitations of microbubbles. A key difference is that the creation of microbubbles is initiated by exposure to ultrasound rather than by activation during injection. The droplets retain their sub-micrometer size while in the bloodstream, enabling them to circulate for longer and potentially to extravasate (e.g., in the leaky vasculature surrounding a tumour [Fig. 2]). Both imaging and therapeutic delivery have been demonstrated using droplets in animal models for multiple applications (
      • Kripfgans O.D.
      • Fowlkes J.B.
      • Miller D.L.
      • Eldevik O.P.
      • Carson P.L.
      Acoustic droplet vaporization for therapeutic and diagnostic applications.
      ;
      • Rapoport N.Y.
      • Gao Z.
      • Kennedy A.
      Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy.
      ;
      • Sheeran P.
      • Luois S.
      • Dayton P.A.
      • Matsunaga T.
      Formulation and acoustic studies of new phase-shift agent for diagnostic and therapeutic ultrasound.
      ). Droplets also offer potential advantages for more recent imaging techniques such as super-resolution by enabling localized activation and destruction; thereby reducing the need for high microbubble concentrations that both degrade the image and pose a potential safety risk (
      • Zhang G.
      • Harput S.
      • Lin S.
      • Christensen-Jeffries K.
      • Leow C.H.
      • Brown J.
      • Dunsby C.
      • Eckersley R.J.
      • Tang M.X.
      Acoustic wave sparsely activated localization microscopy (AWSALM): Super-resolution ultrasound imaging using acoustic activation and deactivation of nanodroplets.
      ). Apart from the phase of the core, their composition is very similar to that of microbubbles and indeed it has been demonstrated that stable droplets can be generated through condensation of commercial perfluorocarbon microbubbles (
      • Sheeran P.S.
      • Yoo K.
      • Williams R.
      • Yin M.
      • Foster F.S.
      • Burns P.N.
      More than bubbles: Creating phase-shift droplets from commercially available ultrasound contrast agents.
      ). The main disadvantages of droplets are that, unlike microbubbles, they cannot be imaged before vaporisation using ultrasound. In addition, relatively high pressures and/or long pulses are typically required to initiate vaporisation. One approach to reducing the requirement for high pressures has been to combine microbubbles and droplets into “acoustic clusters.” It is hypothesized that the shockwave produced by the inertial collapse of the microbubbles at moderate pressure amplitudes facilitates droplet vaporization (
      • Sontum P.
      • Kvåle S.
      • Healey A.J.
      • Skurtveit R.
      • Watanabe R.
      • Matsumura M.
      • Østensen J.
      Acoustic cluster therapy (ACT)— A novel concept for ultrasound mediated, targeted drug delivery.
      ).
      Fig 2
      Fig. 2Overlays of contrast-specific CPS (green) and B-mode (gray) ultrasound scans of HUVEC cells incubated with targeted PCNs: Before activation, no contrast-specific echogenicity is detected, suggesting no microbubbles have formed. After exposure to a mechanical index of 1.1 at 8 MHz, targeted droplets vaporize to the highly echogenic microbubble state and remain in the plane of the HUVEC cells. (Reprinted with permission from
      • Sheeran P.S.
      • Streeter J.E.
      • Mullin L.
      • Matsunaga T.O.
      • Dayton P.A.
      Ultrasound molecular imaging with customizable nanoscale phase-change contrast agents: An in-vitro feasibility study.
      . Copyright 2012, IEEE.)

      Solid cavitation nuclei

      The significant difference between the theoretical tensile strength of a liquid and the pressures at which cavitation is observed has long been attributed to the presence of particles capable of stabilising pockets of gas within hydrophobic cavities (
      • Atchley A.A.
      • Prosperetti A.
      The crevice model of bubble nucleation.
      ). Consequently, a variety of solid particles has also been investigated as agents for bubble generation, primarily for therapeutic applications. These have included particles already used in other applications, such as carbon nanotubes (
      • Delogu L.G.
      • Vidili G.
      • Venturelli E.
      • Menard-Moyon C.
      • Zoroddu M.A.
      • Pilo G.
      • Nicolussi P.
      • Ligios C.
      • Bedognetti D.
      • Sgarrella F.
      • Manetti R.
      • Bianco A.
      Functionalized multiwalled carbon nanotubes as ultrasound contrast agents.
      ) and mesoporous silica (
      • Zhao Y.
      • Zhu Y.C.
      Synergistic cytotoxicity of low-energy ultrasound and innovative mesoporous silica-based sensitive nanoagents.
      ), and deliberately engineered particles such as polymeric “cups” (
      • Kwan J.J.
      • Myers R.
      • Coviello C.M.
      • Graham S.M.
      • Shah A.R.
      • Stride E.
      • Carlisle R.C.
      • Coussios C.C.
      Ultrasound-propelled nanocups for drug delivery.
      ) and gold cones (
      • Mannaris C.
      • Teo B.M.
      • Seth A.
      • Bau L.
      • Coussios C.
      • Stride E.
      Gas-stabilizing gold nanocones for acoustically mediated drug delivery.
      ). Particles that generate gas bubbles through a chemical reaction that can be triggered at a specific site have also been explored (
      • Kang E.
      • Min H.S.
      • Lee J.
      • Han M.H.
      • Ahn H.J.
      • Yoon I.C.
      • Choi K.
      • Kim K.
      • Park K.
      • Kwon I.C.
      Nanobubbles from gas-generating polymeric nanoparticles: Ultrasound imaging of living subjects.
      ). Similar to liquid droplets, these particles can be in the sub micrometer size range, enabling the use of relatively high concentrations, long circulation times and the potential for extravasation. Moreover, solid particles have been demonstrated to offer improved duration of cavitation activity, potentially because, unlike either microbubbles or droplets, the particles are not destroyed by ultrasound exposure. Unfortunately, they also suffer from the same limitations as droplets because they cannot be imaged before activation and require the use of relatively high ultrasound pressures and/or low frequencies compared with microbubbles (
      • Mannaris C.
      • Bau L.
      • Grundy M.
      • Gray M.
      • Lea-Banks H.
      • Seth A.
      • Teo B.
      • Carlisle R.
      • Stride E.
      • Coussios C.C.
      Microbubbles, nanodroplets and gas-stabilizing solid particles for ultrasound-mediated extravasation of unencapsulated drugs: An exposure parameter optimization study.
      ). Furthermore, they may be more difficult to functionalise than microbubbles or droplets for targeted imaging/therapeutic applications because of the need to maintain sufficient surface hydrophobicity. There are also some safety concerns about the use of certain classes of nanomaterial.

      Other novel agents

      Several other sub-micrometer scale agents have been investigated for both imaging and therapeutic applications. These include echogenic liposomes (ELIPs) (
      • Hitchcock K.E.
      • Caudell D.N.
      • Sutton J.T.
      • Klegerman M.E.
      • Vela D.
      • Pyne-Geithman G.J.
      • Abruzzo T.
      • Cyr P.E.P.
      • Geng Y.J.
      • McPherson D.D.
      • Holland C.K.
      Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model.
      ), “nanobubbles” (
      • Cai W.Bin.
      • Yang H.L.
      • Zhang J.
      • Yin J.K.
      • Yang Y.L.
      • Yuan L.J.
      • Zhang L.
      • Duan Y.Y.
      The optimized fabrication of nanobubbles as ultrasound contrast agents for tumor imaging.
      ) and protein coated gas vesicles extracted from bacteria (
      • Shapiro M.G.
      • Goodwill P.W.
      • Neogy A.
      • Yin M.
      • Foster F.S.
      • Schaffer D.V.
      • Conolly S.M.
      Biogenic gas nanostructures as ultrasonic molecular reporters.
      ). Studies of these agents report similar advantages in terms of circulation time and extravasation potential as phase-change liquid droplets and solid cavitation nuclei but without the high activation pressure thresholds. Some controversy exists in the current literature as to the mechanisms of contrast enhancement by these agents (
      • Raymond J.L.
      • Luan Y.
      • Peng T.
      • Huang S.L.
      • McPherson D.D.
      • Versluis M.
      • de Jong N.
      • Holland C.K.
      Loss of gas from echogenic liposomes exposed to pulsed ultrasound.
      ;
      • Hernandez C.
      • Nieves L.
      • de Leon A.C.
      • Advincula R.
      • Exner A.A.
      Role of surface tension in gas nanobubble stability under ultrasound.
      ). They are theoretically too small to resonate at the imaging frequencies typically used in human imaging, and ELIPs and nanobubbles must therefore be used in significantly higher concentrations to produce comparable contrast-to-noise ratios (
      • Kopechek J.A.
      • Haworth K.J.
      • Raymond J.L.
      • Mast T.D.
      • Perrin S.R.
      • Klegerman M.E.
      • Huang S.L.
      • Porter T.M.
      • McPherson D.D.
      • Holland C.K.
      Acoustic characterization of echogenic liposomes: Frequency-dependent attenuation and backscatter.
      ). Moreover, it is an open question whether intrinsic differences exist among ELIPs, nanobubbles and the large quantity of sub-micrometer particles present within a microbubble contrast agent suspension. This controversy is as yet unresolved and, given the currently poor understanding of how bubble populations evolve in a biologic environment and/or after ultrasound exposure, a detailed discussion is outside the scope of this review. Interestingly, the concentrations reported for contrast enhancement by bacterial gas vesicles are significantly lower than for nanobubbles, and further investigation is required to explain this apparent discrepancy. A further potentially important feature of these vesicles is their potential to be engineered to enable cell labelling (e.g., for tracking of micro-organisms in vivo) (
      • Bourdeau R.W.
      • Lee-Gosselin A.
      • Lakshmanan A.
      • Farhadi A.
      • Kumar S.R.
      • Nety S.P.
      • Shapiro M.G.
      Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts.
      ).

      Microbubble Stability

      Static

      Microbubbles tend to thermodynamic equilibrium by converting from a particle suspension to completely separate gas and liquid phases. This process happens by two mechanisms: coalescence and dissolution. The outer coating or shell inhibits the rates of both processes by imposing repulsive surface forces and diminishing surface tension (
      • Borden M.A.
      • Song K.H.
      Reverse engineering the ultrasound contrast agent.
      ), thereby kinetically trapping the microbubbles and stabilizing them for a useful time period. Thus, static microbubbles are relatively stable when the surrounding aqueous medium is saturated with the encapsulated gas (
      • Duncan P.B.
      • Needham D.
      Test of the Epstein-Plesset model for gas microparticle dissolution in aqueous media: Effect of surface tension and gas undersaturation in solution.
      ). In the sealed vial, which is open to energy (heat) transfer but closed to mass transfer, a microbubble suspension can be stable on the shelf for days to months. Once the vial is opened, however, exchange of mass with the atmosphere tends to destabilize the microbubbles, leading to ripening and a decrease in concentration. Indeed, static microbubbles are highly unstable to a degassed medium (
      • Borden M.A.
      • Longo M.L.
      Dissolution behavior of lipid monolayer-coated, air-filled microbubbles: Effect of lipid hydrophobic chain length.
      ) or to gas exchange between the bubble cores and the surrounding milieu (
      • Kwan J.J.
      • Borden M.A.
      Microbubble dissolution in a multigas environment.
      ;
      • Kwan J.J.
      • Borden M.A.
      Lipid monolayer mechanics during microbubble gas exchange.
      ). In general, microbubbles with stiffer, less permeable shells, such as those comprising long acyl-chain lengths, tend to yield more stable microbubbles. The role of emulsifiers is also important in this regard (
      • 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.
      ;
      • Borden M.A.
      Intermolecular forces model for lipid microbubble shells.
      ).

      Acoustic

      Microbubbles that are otherwise stable under static conditions can be altered and destroyed by exposure to ultrasound. Two mechanisms have been identified for acoustic destruction of microbubbles: dissolution and fragmentation (
      • Chomas J.E.
      • Dayton P.A.
      • May D.
      • Allen J.
      • Klibanov A.
      • Ferrara K.
      Optical observation of contrast agent destruction.
      ). Fragmentation is the acoustically driven breakup of a bubble into two or more daughter bubbles (
      • Chomas J.E.
      • Dayton P.
      • Allen J.
      • Morgan K.
      • Ferrara K.W.
      Mechanisms of contrast agent destruction.
      ). High-speed video microscopy has demonstrated that fragmentation likely occurs owing to harmonic shape oscillations (
      • Postema M.
      • Van Wamel A.
      • Lancee C.T.
      • De Jong N.
      Ultrasound-induced encapsulated microbubble phenomena.
      ;
      • Dollet B.
      • Van Der Meer S.
      • Garbin V.
      • De Jong N.
      • Lohse D.
      • Versluis M.
      Nonspherical oscillations of ultrasound contrast agent microbubbles.
      ). Acoustic dissolution, on the other hand, is a process whereby the microbubble diameter shrinks with each pulse, sometimes stabilizing against further acoustically driven dissolution at a small size (1–2 µm diameter) (
      • Borden M.A.
      • Kruse D.E.
      • Caskey C.F.
      • Zhao S.
      • Dayton P.A.
      • Ferrara K.W.
      Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction.
      ). Mechanisms proposed for acoustic dissolution include lipid shedding and fragmentation of a very small daughter bubble (
      • O'Brien J.-P.
      • Ovenden N.
      • Stride E.
      Accounting for the stability of microbubbles to multi-pulse excitation using a lipid-shedding model.
      ;
      • Cox D.J.
      • Thomas J.L.
      Rapid shrinkage of lipid-coated bubbles in pulsed ultrasound.
      ;
      • Thomas D.H.
      • Butler M.
      • Pelekasis N.
      • Anderson T.
      • Stride E.
      • Sboros V.
      The acoustic signature of decaying resonant phospholipid microbubbles.
      ). The onset of these instabilities occurs with increasing mechanical index (MI). Microbubbles are stable when insonified at a very low MI, but they become unstable to acoustic dissolution and then fragmentation as the MI is increased (
      • Chomas J.E.
      • Dayton P.
      • Allen J.
      • Morgan K.
      • Ferrara K.W.
      Mechanisms of contrast agent destruction.
      ). Of note, microbubbles may also attract one another through secondary radiation force (
      • Leighton T.G.
      The acoustic bubble.
      ;
      • Kokhuis T.J.
      • Garbin V.
      • Kooiman K.
      • Naaijkens B.A.
      • Juffermans L.J.
      • Kamp O.
      • van der Steen A.F.
      • Versluis M.
      • de Jong N.
      Secondary bjerknes forces deform targeted microbubbles.
      ), and then coalesce under insonification (
      • Postema M.
      • Marmottant P.
      • Lancée C.T.
      • Hilgenfeldt S.
      • De Jong N.
      Ultrasound-induced microbubble coalescence.
      ).

      In vivo

      Microbubbles injected into the bloodstream are unstable and circulate for only a few minutes before being cleared. Two primary mechanisms have been identified for microbubble clearance: dissolution and phagocytosis. Upon being injected into the bloodstream, freely circulating microbubbles exchange their gas core with the respiratory gases (for the most part N2, O2 and CO2) (
      • Kabalnov A.
      • Klein D.
      • Pelura T.
      • Schutt E.
      • Weers J.
      Dissolution of multicomponent microbubbles in the bloodstream: 1. Theory.
      ) and then completely dissolve, owing to ventilation/perfusion mismatch (
      • Mullin L.
      • Gessner R.
      • Kwan J.
      • Kaya M.
      • Borden M.A.
      • Dayton P.A.
      Effect of anesthesia carrier gas on in vivo circulation times of ultrasound microbubble contrast agents in rats.
      ). In addition, freely circulating microbubbles may be tagged by complement proteins (e.g., C3 b) or other opsonins and then engulfed by phagocytic cells (e.g., macrophages, neutrophils, Kupffer cells). Microbubble phagocytosis has been observed to occur primarily in the lung, liver and spleen (
      • Tartis M.S.
      • Kruse D.E.
      • Zheng H.
      • Zhang H.
      • Kheirolomoom A.
      • Marik J.
      • Ferrara K.W.
      Dynamic microPET imaging of ultrasound contrast agents and lipid delivery.
      ). Of note, phagocytosed microbubbles, or those adhered to the endothelium through ligand-receptor bonds, are more stable than freely circulating microbubbles (
      • Dayton P.A.
      • Rychak J.J.
      Molecular ultrasound imaging using microbubble contrast agents.
      ), perhaps owing to the steadier dissolved gas content in the surrounding microenvironment.

      Targeted Agents

      Microbubbles, as well as droplets and solid particles, can be targeted to a specific cell phenotype, such as inflamed or angiogenic endothelium, by decorating their surfaces with ligand molecules that bind avidly and specifically to a target receptor molecule (e.g., selectins or integrins). Collision of the microbubble with the target cell leads to multiple ligand-receptor interactions, which in turn arrests microbubble motion and adheres it to the cell surface. This enables ultrasound molecular imaging (
      • Dayton P.A.
      • Rychak J.J.
      Molecular ultrasound imaging using microbubble contrast agents.
      ), whereby a combination of pulse sequencing and image analysis is used to detect, visualize and measure the extent of microbubble binding. This method is amenable for receptor molecules that are expressed on the luminal surface of the vascular endothelium. Recently, human clinical trials of ultrasound molecular imaging for detecting tumour angiogenesis were reported for peptide-bearing microbubbles (BR55, Bracco Suisse SA, Geneva, Switzerland) targeted to vascular endothelial growth factor receptor 2 (
      • 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.
      ). In addition, targeted microbubbles may be used to facilitate targeted drug delivery by incorporating chemical specificity in addition to the spatial specificity afforded by ultrasound focusing.

      Surface modification

      In most cases, the targeting ligand is bound to the microbubble surface via a poly(ethylene glycol) (PEG) tether (
      • Klibanov A.L.
      Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging.
      ). The flexible PEG tether allows the ligand molecule to diffuse and orient itself to interact favorably with the receptor binding pocket (
      • Kim D.H.
      • Klibanov A.L.
      • Needham D.
      The influence of tiered layers of surface-grafted poly(ethylene glycol) on receptor-ligand-mediated adhesion between phospholipid monolayer-stabilized microbubbles and coated glass beads.
      ). In some cases, the ligand is attached to the lipid before microbubble production, as is the case for BR55. However, much of the ligand may be wasted by this approach as with some manufacturing techniques, as little as 10% of the precursor lipid used to generate microbubbles incorporates into the microbubble shell. In addition, the targeted microbubbles must be “washed” by centrifugation and resuspension to remove free ligand molecules and thereby prevent competitive binding and blocking of the receptor molecules (
      • Stieger S.M.
      • Dayton P.A.
      • Borden M.A.
      • Caskey C.F.
      • Griffey S.M.
      • Wisner E.R.
      • Ferrara K.W.
      Imaging of angiogenesis using Cadence contrast pulse sequencing and targeted contrast agents.
      ). Conjugation chemistries have therefore been developed to attach the ligand directly onto preformed microbubbles (
      • Klibanov A.L.
      Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents for targeted molecular imaging.
      ). The microbubbles can be washed to clear out free functional groups not attached to the microbubble surface, thereby increasing conjugation yield and reducing cost. Biotin-Avidin-Biotin conjugation chemistry was one of the first approaches to be tested and commercialized (Targestar, Targeson Inc, San Diego, CA, USA; Micromarker, Bracco Suisse SA). In this method, the microbubbles are generated composing PEG-biotin, then decorated with avidin and finally conjugated to a biotinylated ligand (typically an antibody). Unfortunately, avidin is immunogenic, which limits the potential of this approach for clinical translation. Therefore, covalent conjugation chemistries with biocompatible linkers have been developed (
      • Klibanov A.L.
      Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents for targeted molecular imaging.
      ), most notably maleimide-thiol (
      • Geers B.
      • Lentacker I.
      • Sanders N.N.
      • Demeester J.
      • Meairs S.
      • De Smedt S.C.
      Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery.
      ). In this approach, microbubbles formed with PEG-maleimide are washed and then conjugated to thiolated ligand molecules, such as proteins that make up exposed cysteine groups. Unfortunately, this scheme has unwanted side reactions in aqueous media, and exposed maleimide groups after conjugation must be “capped” by free cysteine to prevent nonspecific binding to blood proteins. Recently, biorthogonal click chemistry has been developed to conjugate ligand molecules to microbubbles rapidly and efficiently, thereby removing the need to cap unreacted functional groups on the microbubble surface (
      • Slagle C.J.
      • Thamm D.H.
      • Randall E.K.
      • Borden M.A.
      Click conjugation of cloaked peptide ligands to microbubbles.
      ).

      Buried ligand

      An often-overlooked concern with targeted microbubbles is the potential for complement protein fixation onto the microbubble surface and complement activation of the immune system. Complement protein C3 b is ubiquitous and has an unstable thioester group that can covalently bind to nucleophilic groups on the targeting ligand, such as hydroxyls (
      • Janssen K.G.H.
      • Li J.
      • Hoang H.T.
      • Tas N.R.
      • Van Der Linden H.J.
      • Hankemeier T.
      Downscaling quantitative isotachophoresis: Limits at the sub-picoliter scale.
      ). This means that virtually all targeting ligands can be tagged with C3 b, which has corresponding receptors (C3 R) on nearly every cell of the mononuclear phagocyte system. Microbubble capture and phagocytosis reduce circulation persistence and target specificity, rendering the targeting strategy unreliable unless the target phenotype is inflammation or ischemia. Most ultrasound molecular imaging studies have ignored this important biologic phenomenon, or implicitly assumed that complement fixation is kinetically slow enough in comparison with the microbubble pharmacokinetics and desired ligand-receptor interaction. This assumption is problematic, however, because results have demonstrated that complement fixation of microbubbles can occur in less than 5 min (
      • Borden M.A.
      • Sarantos M.R.
      • Stieger S.M.
      • Simon S.I.
      • Ferrara K.W.
      • Dayton P.A.
      Ultrasound radiation force modulates ligand availability on targeted contrast agents.
      ;
      • Chen C.C.
      • Borden M.A.
      The role of poly(ethylene glycol) brush architecture in complement activation on targeted microbubble surfaces.
      ), and this can significantly reduce microbubble circulation persistence (
      • Chen C.C.
      • Sirsi S.R.
      • Homma S.
      • Borden M.A.
      Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles.
      ). Complement activation can also lead to hypersensitivity reaction that may be fatal (
      • Szebeni J.
      Complement activation-related pseudoallergy: A stress reaction in blood triggered by nanomedicines and biologicals.
      ). Therefore, a buried-ligand approach has been developed to block complement C3 b fixation, prolong microbubble circulation persistence and preserve the microbubble targeting specificity (
      • Borden M.A.
      • Sarantos M.R.
      • Stieger S.M.
      • Simon S.I.
      • Ferrara K.W.
      • Dayton P.A.
      Ultrasound radiation force modulates ligand availability on targeted contrast agents.
      ;
      • Borden M.A.
      • Zhang H.
      • Gillies R.J.
      • Dayton P.A.
      • Ferrara K.W.
      A stimulus-responsive contrast agent for ultrasound molecular imaging.
      ;
      • Borden M.A.
      • Streeter J.E.
      • Sirsi S.R.
      • Dayton P.A.
      In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.
      ). The approach employs a tiered PEG brush layer with the ligand tethered by short PEG chains and surrounded by methyl-terminated long PEG chains that protect the ligand against complement attack. The ligand is then revealed for binding to the receptor by the primary ultrasound radiation force.

      Magnetic targeting

      An alternative and potentially complementary targeting approach is to encapsulate magnetic material within the microbubble coating, thereby making the microbubbles responsive to an externally applied magnetic field. Magnetic targeting of microbubbles has been demonstrated to enable enhanced delivery of multiple therapeutics in vitro and in vivo (
      • Stride E.
      • Porter C.
      • Prieto A.G.
      • Pankhurst Q.
      Enhancement of microbubble mediated gene delivery by simultaneous exposure to ultrasonic and magnetic fields.
      ;
      • Vlaskou D.
      • Mykhaylyk O.
      • Pradhan P.
      • Bergemann C.
      • Klibanov A.L.
      • Hensel K.
      • Schmitz G.
      • Plank C.
      Magnetic microbubbles: Magnetically targeted and ultrasound-triggered vectors for gene delivery in vitro.
      ), accelerated thrombolysis (
      • de Saint Victor M.
      • Barnsley L.C.
      • Carugo D.
      • Owen J.
      • Coussios C.C.
      • Stride E.
      Sonothrombolysis with magnetically targeted microbubbles.
      ), improved contrast enhancement and to provide magnetic resonance image contrast enhancement (
      • Crake C.
      • Owen J.
      • Smart S.
      • Coviello C.
      • Coussios C.C.
      • Carlisle R.
      • Stride E.
      Enhancement and passive acoustic mapping of cavitation from fluorescently tagged magnetic resonance-visible magnetic microbubbles in vivo.
      ).

      Theranostic Agents

      The term “theranostic” was originally coined to describe screening techniques to predict a patient's suitability for a particular treatment (
      • Pene F.
      • Courtine E.
      • Cariou A.
      • Mira J.-P.
      Toward theragnostics.
      ). Its meaning has been extended, however, to describe methods and technologies that integrate diagnostic and therapeutic procedures. Microbubbles have multiple features that make them highly effective as theranostic agents. Their echogenicity provides a convenient means of real time treatment monitoring because both their location and amplitude of oscillation can be determined using conventional ultrasound imaging equipment. The same oscillatory behavior that makes microbubbles echogenic, also gives rise to several biological effects that can be exploited for therapeutic purposes. Specifically, when a microbubble or cloud of microbubbles oscillate, the surrounding fluid can be set into motion. This “microstreaming” can significantly enhance convection of drugs from the bloodstream into tissue (
      • Mannaris C.
      • Bau L.
      • Grundy M.
      • Gray M.
      • Lea-Banks H.
      • Seth A.
      • Teo B.
      • Carlisle R.
      • Stride E.
      • Coussios C.C.
      Microbubbles, nanodroplets and gas-stabilizing solid particles for ultrasound-mediated extravasation of unencapsulated drugs: An exposure parameter optimization study.
      ). This is particularly important for the treatment of solid tumours and blood clots where diffusion is often insufficient to produce therapeutic drug concentrations throughout the target tissue. Microstreaming is also thought to be one of the mechanisms by which microbubbles can promote reversible opening of the endothelium, including the blood–brain barrier and enhance cellular uptake of drugs (sonoporation). To date, clinical studies have utilized existing commercial contrast agents (
      • Carpentier A.
      • Canney M.
      • Vignot A.
      • Reina V.
      • Beccaria K.
      • Horodyckid C.
      • Karachi C.
      • Leclercq D.
      • Lafon C.
      • Chapelon J.Y.
      • Capelle L.
      • Cornu P.
      • Sanson M.
      • Hoang-Xuan K.
      • Delattre J.Y.
      • Idbaih A.
      Clinical trial of blood-brain barrier disruption by pulsed ultrasound.
      ), but pre-clinical studies have demonstrated that therapeutic efficacy can be enhanced by refining the microbubble agent size distribution (
      • Chen H.
      • Konofagou E.E.
      The size of blood-brain barrier opening induced by focused ultrasound is dictated by the acoustic pressure.
      ) or exploiting the same targeting strategies as described in the previous section of this report to increase microbubble concentration at the treatment site. At higher ultrasound pressures, microbubbles and their precursors can also produce both mechanical and thermal ablation of tissue. For a more detailed review of therapeutic applications please see the companion review in this special issue of Ultrasound in Medicine and Biology by Kooiman et al. (2020).
      Microbubble agents can also be used to localize the release and/or activation of drugs. For example,
      • Bezagu M.
      • Clarhaut J.
      • Renoux B.
      • Monti F.
      • Tanter M.
      • Tabeling P.
      • Cossy J.
      • Couture O.
      • Papot S.
      • Arseniyadis S.
      In situ targeted activation of an anticancer agent using ultrasound-triggered release of composite droplets.
      utilized a specially designed form of composite droplet to encapsulate a prodrug of chemotherapeutic agent and demonstrated that its release was confined to the droplet activation site. Certain classes of drug become functional only when exposed to a physical stimulus. Their activity can thus be confined to a target tissue volume, which significantly reduces the risk of systemic toxicity. It has been demonstrated that ultrasound and microbubbles can provide a suitable stimulus (
      • Yumita N.
      • Nishigaki R.
      • Umemura K.
      • Umemura S.
      Hematoporphyrin as a sensitizer of cell‐damaging effect of ultrasound.
      ;
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ) to enable so-called sonodynamic therapy. The underpinning mechanisms are still the subject of some debate but may relate to the production of light by oscillating microbubbles (sonoluminescence) (
      • Beguin E.
      • Shrivastava S.
      • Dezhkunov N.V.
      • McHale A.P.
      • Callan J.F.
      • Stride E.
      Direct evidence of multibubble sonoluminescence using therapeutic ultrasound and microbubbles.
      ).
      Microbubbles can be used for the delivery of therapeutic gases such as oxygen and nitric oxide (
      • Sutton J.T.
      • Raymond J.L.
      • Verleye M.C.
      • Pyne-Geithman G.J.
      • Holland C.K.
      Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue.
      ;
      • McEwan C.
      • Owen J.
      • Stride E.
      • Fowley C.
      • Nesbitt H.
      • Cochrane D.
      • Coussios C.C.
      • Borden M.
      • Nomikou N.
      • McHale A.P.
      • Callan J.F.
      Oxygen carrying microbubbles for enhanced sonodynamic therapy of hypoxic tumours.
      ), and their outer coating also provides a versatile platform for attachment or encapsulation of drug molecules. This facilitates targeted release because microbubbles can be stimulated to release the drug at the target site using focused ultrasound. This, combined with the enhanced tissue penetration discussed earlier in this report, typically also significantly reduces the quantity of drug that needs to be injected. A detailed review of the various conjugation/encapsulation methods may be found in
      • 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.
      . An overview summary is presented in Figure 3.
      Fig 3
      Fig. 3Conjugation strategies for microbubbles agents used for molecular imaging and/or therapeutic delivery applications. (Reproduced with permission from
      • 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.
      . Copyright 2017, IEEE.)

      Multi-Modality Imaging Agents

      Microbubbles can also be functionalized to enable them to be visualized with other imaging techniques to facilitate multi-modality imaging (Fig. 4).
      Fig 4
      Fig. 4Examples of multi-modality imaging of microbubbles. (a) Photoacoustic image of gold nanoparticles conjugated to lipid microbubbles within a murine tumour. (Reproduced with permission from
      • Meng Z.
      • Zhou X.
      • She J.
      • Zhang Y.
      • Feng L.
      • Liu Z.
      Ultrasound-responsive conversion of microbubbles to nanoparticles to enable background-free in vivo photoacoustic imaging.
      . Copyright 2019, American Chemical Society.) (b) MR image of murine tumour after injection of lipid microbubbles containing iron oxide nanoparticles. (Reproduced with permission from
      • Crake C.
      • Owen J.
      • Smart S.
      • Coviello C.
      • Coussios C.C.
      • Carlisle R.
      • Stride E.
      Enhancement and passive acoustic mapping of cavitation from fluorescently tagged magnetic resonance-visible magnetic microbubbles in vivo.
      . Copyright 2016, Elsevier). (c) Time series of phase contrast X-ray images of tube containing a suspension of polymeric microbubbles with diminishing concentration. (Reproduced with permission from
      • Millard T.P.
      • Endrizzi M.
      • Everdell N.
      • Rigon L.
      • Arfelli F.
      • Menk R.H.
      • Stride E.
      • Olivo A.
      Evaluation of microbubble contrast agents for dynamic imaging with X-ray phase contrast.
      . Copyright 2015, Springer Nature.) (d) Maximum intensity projection PET image of a mouse after injection of radiolabelled microbubbles. (Reproduced with permission from
      • Tartis M.S.
      • Kruse D.E.
      • Zheng H.
      • Zhang H.
      • Kheirolomoom A.
      • Marik J.
      • Ferrara K.W.
      Dynamic microPET imaging of ultrasound contrast agents and lipid delivery.
      . Copyright 2008, Elsevier B.V.)

      Optical

      Particularly for in vitro and pre-clinical studies, it is often beneficial to visualize microbubbles with optical microscopy, using a fluorescent dye molecule incorporated into the microbubble shell (e.g., intravital microscopy to view microbubble circulation dynamics) (
      • Unger E.
      • Porter T.
      • Lindner J.
      • Grayburn P.
      Cardiovascular drug delivery with ultrasound and microbubbles.
      ). Dye molecules can be conjugated onto the microbubble surface (
      • Upadhyay A.
      • Dalvi S.V.
      • Gupta G.
      • Khanna N.
      Effect of PEGylation on performance of protein microbubbles and its comparison with lipid microbubbles.
      ;
      • Slagle C.J.
      • Thamm D.H.
      • Randall E.K.
      • Borden M.A.
      Click conjugation of cloaked peptide ligands to microbubbles.
      ), or they can be loaded into the microbubble shell (
      • Klibanov A.L.
      Ultrasound contrast agents: Development of the field and current status.
      ). Lipophilic cell labeling dyes (DiI, DiO, DiD, etc.; Vybrant, Thermo-Fisher, Waltham, MA, USA) are particularly useful for tagging lipid-coated microbubbles. Fluorescent molecules can quench their fluorescence at high concentration, so one must be cautious to avoid overloading the microbubble with dye. Some dyes can also photo-quench more rapidly than others, so it is useful to investigate photo-stability during the microbubble design phase. Fluorescent nanoparticle dyes, such as quantum dots (
      • Ke H.
      • Xing Z.
      • Zhao B.
      • Wang J.
      • Liu J.
      • Guo C.
      • Yue X.
      • Liu S.
      • Tang Z.
      • Dai Z.
      Quantum-dot-modified microbubbles with bi-mode imaging capabilities.
      ), have also been conjugated to microbubbles to enhance optical visualization. Optical coherence tomography produces images from reflected light, using interferometry to reject multiple scattering. Similar to ultrasound imaging, contrast can be improved by the introduction of strong scatterers such as microbubbles (
      • Lee T.M.
      • Oldenburg A.L.
      • Sitafalwalla S.
      • Marks D.L.
      • Luo W.
      • Toublan F.J.-J.
      • Suslick K.S.
      • Boppart S.A.
      Engineered microsphere contrast agents for optical coherence tomography.
      ). Methods such as ultrasound-modulated fluorescence have also been developed to combine optical and ultrasound imaging to provide fluorescence contrast at ultrasound resolution in optically scattering media (
      • Liu Y.
      • Feshitan J.A.
      • Wei M.-Y.
      • Borden M.A.
      • Yuan B.
      Ultrasound-modulated fluorescence based on fluorescent microbubbles.
      ).

      Photoacoustics

      In addition, optically absorbing plasmonic gold nanoparticles can be conjugated onto microbubbles for dual modality ultrasound and photoacoustic imaging (
      • Dove J.D.
      • Murray T.W.
      • Borden M.A.
      Enhanced photoacoustic response with plasmonic nanoparticle-templated microbubbles.
      ). Plasmonic nanoparticles absorb photons and dissipate heat, leading to localized photo-thermal expansion and production of an acoustic wave. Such pulsed heating can also drive microbubble oscillations when the plasmonic nanoparticles are arrayed around a microbubble gas core (
      • Dove J.D.
      • Borden M.A.
      • Murray T.W.
      Optically induced resonance of nanoparticle-loaded microbubbles.
      ). Gold nanoparticle-coated microbubbles focus the photoacoustic response at the microbubble resonance frequency, rather than the usual broadband response, thereby providing a more efficient energy conversion for sensing with medical ultrasound probes. Gold nanoparticles can also be loaded into phase-change emulsion droplets, where vaporization leads to an enhanced photoacoustic effect (
      • Wilson K.
      • Homan K.
      • Emelianov S.
      Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging.
      ;
      • Meng Z.
      • Zhou X.
      • She J.
      • Zhang Y.
      • Feng L.
      • Liu Z.
      Ultrasound-responsive conversion of microbubbles to nanoparticles to enable background-free in vivo photoacoustic imaging.
      ) (Fig. 4a). The vaporization event can be tailored to specific optical wavelengths, such as the near infrared, by selection of the nanoparticle structure (
      • Dixon A.J.
      • Hu S.
      • Klibanov A.L.
      • Hossack J.A.
      Oscillatory dynamics and in vivo photoacoustic imaging performance of plasmonic nanoparticle-coated microbubbles.
      ) or to laser fluence by choice of the fluorocarbon core (
      • Dove J.D.
      • Mountford P.A.
      • Murray T.W.
      • Borden M.A.
      Engineering optically triggered droplets for photoacoustic imaging and therapy.
      ). Alternatively, the microbubbles can be coated with a dye-loaded liquid layer (
      • Lajoinie G.
      • Lee J.-Y.
      • Owen J.
      • Kruizinga P.
      • de Jong N.
      • van Soest G.
      • Stride E.
      • Versluis M.
      Laser-driven resonance of dye-doped oil-coated microbubbles: Experimental study.
      ), which offers both an easier way to tune the absorption wavelength and removes the long-term biocompatibility concerns of gold nanoparticles.

      Magnetic resonance imaging

      Microbubbles can be used as contrast agents for magnetic resonance imaging (MRI) without modification because of their ability to produce a difference in magnetic susceptibility when injected into the bloodstream (
      • Ueguchi T.
      • Tanaka Y.
      • Hamada S.
      • Kawamoto R.
      • Ogata Y.
      • Matsumoto M.
      • Nakamura H.
      • Johkoh T.
      Air microbubbles as MR susceptibility contrast agent at 1.5 tesla.
      ). To increase the contrast-to-noise ratio, microbubbles can also be loaded with hyperpolarized gases (
      • Callot V.
      • Canet E.
      • Brochot J.
      • Viallon M.
      • Humblot H.
      • Briguet A.
      • Tournier H.
      • Crémillieux Y.
      MR perfusion imaging using encapsulated laser-polarized 3 He.
      ) or particulate MRI contrast agents for dual modality imaging. In one example, polymer shelled microbubbles were loaded with the T2 contrast agent, iron oxide (
      • Yang F.
      • Li Y.
      • Chen Z.
      • Zhang Y.
      • Wu J.
      • Gu N.
      Superparamagnetic iron oxide nanoparticle-embedded encapsulated microbubbles as dual contrast agents of magnetic resonance and ultrasound imaging.
      ). In another example, iron oxide particles were incorporated into the shell of lipid-coated microbubbles (
      • Owen J.
      • Crake C.
      • Lee J.Y.
      • Carugo D.
      • Beguin E.
      • Khrapitchev A.A.
      • Browning R.J.
      • Sibson N.
      • Stride E.
      A versatile method for the preparation of particle-loaded microbubbles for multimodality imaging and targeted drug delivery.
      ) (Fig. 4b). The T1 contrast agent, Gd(III), has also been loaded onto microbubbles with polymer shells (
      • Ao M.
      • Wang Z.
      • Ran H.
      • Guo D.
      • Yu J.
      • Li A.
      • Chen W.
      • Wu W.
      • Zheng Y.
      Gd-DTPA-loaded PLGA microbubbles as both ultrasound contrast agent and MRI contrast agent—A feasibility research.
      ) or lipid shells (
      • Feshitan J.A.
      • Boss M.A.
      • Borden M.A.
      Magnetic resonance properties of Gd(III)-bound lipid-coated microbubbles and their cavitation fragments.
      ). Of note,
      • Feshitan J.A.
      • Vlachos F.
      • Sirsi S.R.
      • Konofagou E.E.
      • Borden M.A.
      Theranostic Gd(III)-lipid microbubbles for MRI-guided focused ultrasound surgery.
      found that Gadoteric acid (Gd-DOTA) did not enhance the T1 image for the intact microbubble, but it did enhance the signal for a fragmented bubble. This raised the tantalizing possibility of using MRI to guide ultrasound-targeted microbubble destruction, but unfortunately the signal-to-noise ratio has been too low for in vivo validation. Owing to the growing interest in MRI-guided focused ultrasound therapy, there remains strong interest in novel MRI-detectable microbubbles and nanodrops (
      • Koshkina O.
      • Lajoinie G.
      • Baldelli Bombelli F.
      • Swider E.
      • Cruz L.J.
      • White P.B.
      • Schweins R.
      • Dolen Y.
      • van Dinther E.A.W.
      • van Riessen N.K.
      • Rogers S.E.
      • Fokkink R.
      • Voets I.K.
      • van Eck E.R.H.
      • Heerschap A.
      • Versluis M.
      • de Korte C.L.
      • Figdor C.G.
      • de Vries I.J.M.
      • Srinivas M.
      Multicore liquid perfluorocarbon-loaded multimodal nanoparticles for stable ultrasound and 19 F MRI applied to in vivo cell tracking.
      ).

      X-ray

      Although invisible to conventional X-ray imaging, microbubbles can be loaded with X-ray opaque material to provide contrast. They can also be imaged directly using X-ray phase contrast imaging. Individual microbubbles act as lenses, refracting the incoming X-rays and producing a large phase difference when radiation is propagated through a microbubble population (
      • Millard T.P.
      • Endrizzi M.
      • Rigon L.
      • Arfelli F.
      • Menk R.H.
      • Owen J.
      • Stride E.
      • Olivo A.
      Quantification of microbubble concentration through X-ray phase contrast imaging.
      ) (Fig. 4c). The comparative safety of microbubbles compared with iodine or other X-ray contrast agents makes them a potentially attractive alternative.

      Positron emission tomography/single photon emission computed tomography

      Microbubbles can be radio-labelled and imaged with single photo emission computed tomography or positron emission tomography isotopes to determine bio-distribution. This can be useful for assessing targeting efficiency and impact of acoustic exposure (
      • Tartis M.S.
      • Kruse D.E.
      • Zheng H.
      • Zhang H.
      • Kheirolomoom A.
      • Marik J.
      • Ferrara K.W.
      Dynamic microPET imaging of ultrasound contrast agents and lipid delivery.
      ) (Fig. 4d).

      Microbubble Synthesis

      Sonication

      Sonication was the first method described to generate microbubbles for ultrasonics (
      • Feinstein S.B.
      • Ten Cate F.J.
      • Zwehl W.
      • Ong K.
      • Maurer G.
      • Tei C.
      • Shah P.M.
      • Meerbaum S.
      • Corday E.
      Two-dimensional contrast echocardiography: In vitro development and quantitative analysis of echo contrast agents.
      ). Sonication involves the high-frequency vibration (typically 20 kHz) of a horn tip at the gas/water interface. This vibration leads to gas entrainment and secondary breakup through cavitation in the bulk aqueous phase (
      • Li M.K.
      • Fogler H.S.
      Acoustic emulsification. Part 2. Breakup of the large primary oil droplets in a water medium.
      ,
      • Li M.K.
      • Fogler H.S.
      Acoustic emulsification. Part 1. The instability of the oil-water interface to form the initial droplets.
      ). Unfortunately, there is very little research into the details of gas entrainment via sonication. It is generally known that low-power sonication with the tip submerged inside the aqueous medium leads to microbubble destruction (clarification) and breakup of the lipid structures from multi-lamellar vesicles into small uni-lamellar liposomes. This is often a preparatory step in generating microbubbles. Microbubbles are then generated by moving the probe tip to the gas/water interface and turning the system to full power. Sonication generates many microbubbles very rapidly: for example, a 1 L volume of 1012 bubbles/L can be generated within 1 min. The stochastic processes of entrainment and breakup lead to a fairly polydisperse size distribution, but this can be refined using sorting techniques as outlined later in this report. Thus, sonication is a simple and economic method of generating microbubbles in high yield.

      Shaking

      Shaking is another process of mechanical agitation that is used to create microbubbles. Typically, a small volume (∼1 mL) of lipid solution is sealed in a small vial with a gas headspace and placed in a dental amalgamator or similar mixing device. The device vibrates along the long axis of the vial at ∼4000 Hz. This method is used to generate Definity microbubbles (Lantheus, North Billerica, MA, USA), for example. The benefit of the shaking method is that it can produce microbubbles rapidly (∼109 bubbles in less than 1 min) on demand. The lipid suspension in the vial can be made at a central facility, sterilized and then shipped to the end user, who simply places the vial into the shaking device to generate the microbubbles (e.g., at the bedside). Thus, it is a very simple and economic method for multiple uses of small quantities of microbubbles. As with sonication, the bubble entrainment and breakup processes during shaking are poorly understood, but it is known that they also lead to a polydisperse size distribution. Coincidentally, the size distribution of microbubbles formed by shaking tends to be remarkably similar to that of microbubbles formed by sonication, despite the very different geometry and characteristic frequency. More research is necessary to better understand the bubble formation process during shaking, as well as how the shaking parameters affect the microbubble size distribution.

      Microfluidics

      As above, the standard microbubble production methods result in inherently polydisperse microbubble size distributions, typically ranging from 1–10 µm in diameter (
      • Stride E.
      • Saffari N.
      Microbubble ultrasound contrast agents: A review.
      ). A microbubble of a given size and coating properties will resonate, and thus generate the strongest echo, at a specific frequency (
      • Minnaert M.
      On musical air-bubbles and the sound of running water.
      ). Hence, this polydispersity can be advantageous in the case of a multi-purpose imaging contrast agent used for a range of various anatomic targets and consequently with a range of various imaging frequencies. It does, however, limit the contrast-to-noise ratio that can be achieved for a given microbubble concentration under any one set of imaging conditions. Consequently, monodispersity in terms of size and acoustic response is regarded as an important condition to unlock the full potential of microbubbles both for imaging and therapy by providing the possibility to finely control the bubble response and efficiently make use of their resonance. Moreover, monodisperse populations allow for a more effective use of bubble non-linearities, otherwise drowned in the bubble-to-bubble variations in terms of acoustic response. This is particularly important for novel imaging strategies such as super-resolution, 3-D and plane wave imaging in which bubble concentrations may be restricted and the need to maximise bubble signal at low ultrasound pressures is critical (
      • Forsberg F.
      • Rawool N.M.
      • Merton D.A.
      • Liu J.B.
      • Goldberg B.B.
      Contrast enhanced vascular three-dimensional ultrasound imaging.
      ;
      • Couture O.
      • Fink M.
      • Tanter M.
      Ultrasound contrast plane wave imaging.
      ;
      • Ghosh D.
      • Xiong F.
      • Sirsi S.R.
      • Shaul P.W.
      • Mattrey R.F.
      • Hoyt K.
      Toward optimization of in vivo super-resolution ultrasound imaging using size-selected microbubble contrast agents.
      ;
      • Lin F.
      • Tsuruta J.K.
      • Rojas J.D.
      • Dayton P.A.
      Optimizing sensitivity of ultrasound contrast-enhanced super-resolution imaging by tailoring size distribution of microbubble contrast agent.
      ;
      • Jones R.M.
      • Deng L.L.
      • Leung K.
      • McMahon D.
      • O'Reilly M.A.
      • Hynynen K.
      Three-dimensional transcranial microbubble imaging for guiding volumetric ultrasound-mediated blood-brain barrier opening.
      ). Therefore, monodisperse contrast agents, by offering new imaging possibilities (
      • Segers T.
      • Kruizinga P.
      • Kok M.P.
      • Lajoinie G.
      • de Jong N.
      • Versluis M.
      Monodisperse versus polydisperse ultrasound contrast agents: Non-linear response, sensitivity, and deep tissue imaging potential.
      ), could be considered novel agents in their own right. Furthermore, because of their uniform acoustic response (
      • Segers T.
      • de Jong N.
      • Versluis M.
      Uniform scattering and attenuation of acoustically sorted ultrasound contrast agents: Modeling and experiments.
      ), monodisperse microbubble populations may potentially solve remaining fundamental questions as to the optimal acoustic parameters 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.A.
      • Bera D.
      • Luan Y.
      • van der Steen A.F.W.
      • 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.
      ) and to precisely measure the role of shell components on the acoustic bubble response (
      • Segers T.
      • Gaud E.
      • Versluis M.
      • Frinking P.
      High-precision acoustic measurements of the non-linear dilatational elasticity of phospholipid coated monodisperse microbubbles.
      ). Therefore, throughout the past decades, several techniques have been developed to produce bubble suspensions with a narrow size distribution. The first approach is to enrich polydisperse microbubbles and the second approach is to directly form monodisperse bubbles in a microfluidic flow-focusing device. Microbubbles can be enriched through mechanical filtration over a pore filter (
      • 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.
      ), through decantation (
      • Goertz D.E.
      • de Jong N.
      • van der Steen A.F.W.
      Attenuation and size distribution measurements of Definity and manipulated Definity populations.
      ) and through multiple centrifugation steps (
      • Feshitan J.A.
      • Chen C.C.
      • Kwan J.J.
      • Borden M.A.
      Microbubble size isolation by differential centrifugation.
      ). With a higher degree of control, microbubbles can be sorted in microfluidic devices resulting in even narrower size distributions. They can be sorted to size in a pinched microchannel (
      • Kok M.P.
      • Segers T.
      • Versluis M.
      Bubble sorting in pinched microchannels for ultrasound contrast agent enrichment.
      ) and they can be sorted to their resonance behavior using the primary radiation force induced by a traveling acoustic wave (
      • Segers T.
      • Versluis M.
      Acoustic bubble sorting for ultrasound contrast agent enrichment.
      ). A challenge for microfluidic sorting methods is to increase robustness and throughput and, therefore, to scale up (e.g., through parallelization). An advantage of sorting methods is the freedom of choice in the lipid mixture used to coat the bubbles (i.e., today, the constraints in terms of the formulations for microfluidic bubble formation at high production rates are rather stringent) (

      Hettiarachchi, Kanaka and Talu, Esra and Longo, Marjorie L and Dayton, Paul A and Lee, Abraham P. On-chip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging. 2007;463–468. Royal Society of Chemistry.

      ;
      • 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 flow-focusing.
      ).

      Microfluidic flow-focusing

      The second approach to produce a monodisperse bubble suspension is through direct bubble formation in a microfluidic flow-focusing device in which a gas thread is focused between two liquid co-flows through a constriction where it destabilizes and pinches off to release monodisperse bubbles (Fig. 5). The bubble size and the bubble formation rate in a flow-focusing device can be controlled through the gas pressure and the liquid flow rate. Historically, a large gap has separated the classic large-scale microbubble manufacturing techniques presented earlier in this report and the microfluidic flow-focusing technique, regarded as a low-throughput method. Nevertheless, owing to a better understanding of the microscale fluid dynamics (
      • Ganán-Calvo A.M.
      • Gordillo J.M.
      Perfectly monodisperse microbubbling by capillary flow 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.
      ) and innovative designs (
      • Castro-Hernández E.
      • van Hoeve W.
      • Lohse D.
      • Gordillo J.M.
      Microbubble generation in a co-flow device operated in a new regime.
      ), to date, flow-focusing allows for the formation of several million bubbles per second (
      • Castro-Hernández E.
      • van Hoeve W.
      • Lohse D.
      • Gordillo J.M.
      Microbubble generation in a co-flow 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.
      ), allowing for the production of a clinically relevant dose (i.e., 109 bubbles) in a matter of minutes.
      Fig 5
      Fig. 5(a) Flow-focusing microfluidic chip used to produce monodisperse phospholipid-coated microbubbles. (Reproduced with permission from
      • 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 flow-focusing.
      . Copyright 2017, American Chemical Society.) (b) Microfluidic chip designed to produce multi-layer bubbles. (Reproduced with permission from
      • Hettiarachchi K.
      • Lee A.P.
      • Zhang S.
      • Feingold S.
      • Dayton P.A.
      Controllable microfluidic synthesis of multiphase drug-carrying lipospheres for site-targeted therapy.
      . Copyright 2019, American Institute of Chemical Engineers.) (c) Parallelized microfluidic chip. (Reproduced with permission from
      • Jeong H.H.
      • Yadavali S.
      • Issadore D.
      • Lee D.
      Liter-scale production of uniform gas bubbles via parallelization of flow-focusing generators.
      . Copyright 2017, Royal Society of Chemistry.) (d) SEM image of a silica particle-coated microbubble. (Reproduced with permission from
      • Lee S.S.
      • Park J.W.
      • Pelet S.
      • Hegemann B.
      • Jeon N.L.
      • Peter M.
      Microfluidic-Based Assay Platform for Studying Polarization Mechanism of Budding Yeast Under Gradient of Mating Pheromone.
      . Copyright 2013, Royal Society of Chemistry). (e) Hard shell microbubbles. (Reproduced with permission from
      • Shih C.P.
      • Chen H.C.
      • Chen H.K.
      • Chiang M.C.
      • Sytwu H.K.
      • Lin Y.C.
      • Li S.L.
      • Shih Y.F.
      • Liao A.H.
      • Wang C.H.
      Ultrasound-aided microbubbles facilitate the delivery of drugs to the inner ear via the round window membrane.
      . Copyright 2013, Elsevier B.V.) (f) Lipid-coated microbubble. (Reproduced with permission from
      • Seo M.
      • Matsuura N.
      Mondisperse, submicrometer droplets via condensation of microfluidc-generated gas bubbles.
      . Copyright 2012, Wiley.) (g) Droplet as microbubbles precursors produced using microfluidics. (Reproduced with permission from
      • Abbaspourrad A.
      • Duncanson W.J.
      • Lebedeva N.
      • Kim S.H.
      • Zhushma A.P.
      • Datta S.S.
      • Dayton P.A.
      • Sheiko S.S.
      • Rubinstein M.
      • Weitz D.A.
      Microfluidic fabrication of stable gas-filled microcapsules for acoustic contrast enhancement.
      . Copyright 2013, American Chemical Society.)

      Fluid physics of microfluidic flow-focusing

      Much work has been done on the microscale fluid dynamics that governs the production of microbubbles using microfluidics, including the pinch-off process, gas jet formation and break-up and the effect of the flow-focusing geometry (
      • Ganán-Calvo A.M.
      • Gordillo J.M.
      Perfectly monodisperse microbubbling by capillary flow 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.
      ;
      • 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.
      ;
      • van Hoeve W.
      • Dollet B.
      • Versluis M.
      • Lohse D.
      Microbubble formation and pinch-off scaling exponent in flow-focusing devices.
      ). These investigations, often made in terms of scaling laws, have had a large impact on the capability of microfluidic techniques and led to an improvement of 3 orders-of-magnitude in the production rate of a single microfluidic nozzle. Notwithstanding the importance of these advances, much remains to be done, on the one hand on the fluid dynamics within the chip including the nozzle or constriction to obtain a complete and predictive description of these techniques and, on the other hand, on the impact of the coating material on the production dynamics.

      Long-term size stability of microfluidically formed bubbles

      Microfluidically formed lipid-coated bubbles are inherently unstable and prone to Ostwald ripening until they have dissolved to their stable size which is typically 2–3 times smaller than their initial on-chip bubble size (
      • Talu E.
      • Hettiarachchi K.
      • Powell R.J.
      • Lee A.P.
      • Dayton P.A.
      • Longo M.L.
      Maintaining monodispersity in a microbubble population formed by flow-focusing.
      ;
      • 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.
      ;
      • Shih R.
      • Lee A.P.
      Post-formation shrinkage and stabilization of microfluidic bubbles in lipid solution.
      ). Once the bubbles have reached their final size, they are stable for days in a closed vial (
      • 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 size decrease during stabilization has been characterized and it is a function of the lipid mixture in the sense that it increases proportionally with the molar amount of PEGylated lipids and with the PEG chain length (
      • 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 flow-focusing.
      ). As a result of the size decrease during stabilization, approximately 90% of the initial gas volume diffuses out of the freshly formed bubbles, which first saturates the surrounding liquid and then results in large foam bubbles formed through Ostwald ripening (
      • Talu E.
      • Hettiarachchi K.
      • Powell R.J.
      • Lee A.P.
      • Dayton P.A.
      • Longo M.L.
      Maintaining monodispersity in a microbubble population formed by flow-focusing.
      ;
      • 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.
      ). One major challenge is to investigate how foam formation can be mitigated to produce readily usable monodisperse microbubble suspensions.

      On-chip stability of microfluidically formed bubbles

      Microfluidic monodisperse bubble formation requires lipid concentrations approximately 10 times higher than those in classic microbubble production methods to minimize on-chip bubble coalescence in the outlet of the flow-focusing device (
      • Shih R.
      • Lee A.P.
      Post-formation shrinkage and stabilization of microfluidic bubbles in lipid solution.
      ;
      • 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 flow-focusing.
      ). Furthermore, a high molar concentration of PEGylated lipids with a long chain length is required to minimize coalescence which to date limits the freedom in the choice of lipid mixture for direct monodisperse bubble formation. It has been demonstrated that the stability against on-chip bubble coalescence can be improved by forming bubbles at elevated temperatures (
      • 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.
      ). However, high lipid concentrations are still required, for the most part because of the crucial function of the free liposomes in inhibiting coalescence. A future challenge is therefore to find a more economic and equally effective alternative to these free liposomes, allowing to reduce the concentration used and subsequently, the costs.

      Microbubble uniformity

      Size monodispersity is a critical and necessary aspect to control the acoustic microbubble responses and it can be accurately controlled by the flow-focusing method. It is not, however, sufficient. More precisely, the response of a microbubble depends on its size and it is also strongly affected by the viscoelastic properties of the shell (
      • van der Meer S.
      • Dollet B.
      • Voormolen M.
      • Chin C.T.
      • Bouakaz A.
      • de Jong N.
      • Versluis M.
      • Lohse D.
      Microbubble spectroscopy of ultrasound contrast agents.
      ). Lipid-coated bubbles, for example, represent most ultrasound contrast agents currently under investigation and under clinical use. It has been demonstrated that the response to an ultrasound pulse strongly depends on the lipid packing density (i.e., on the equilibrium surface tension) (
      • Overvelde M.
      • Garbin V.
      • Sijl J.
      • Dollet B.
      • de Jong N.
      • Lohse D.
      • Versluis M.
      Nonlinear shell behavior of phospholipid-coated microbubbles.
      ). A future challenge is to measure the acoustic uniformity of mono-sized bubbles formed by flow-focusing. Furthermore, it remains to be investigated how the acoustic response of lipid-coated bubbles can be tuned (i.e., how various lipid shell components affect the acoustic microbubble response). Control over the viscoelastic shell properties and thus over the acoustic response would be highly valuable because this would allow for microbubble design dedicated to specific clinical applications such as non-invasive blood pressure measurements, sonothrombolysis and drug and gene delivery using bubbles and ultrasound.

      In vivo performance

      It has been demonstrated in an in vitro setup that the use of a monodisperse bubble population results in a sensitivity increase of 2–3 orders of magnitude (
      • Segers T.
      • Kruizinga P.
      • Kok M.P.
      • Lajoinie G.
      • de Jong N.
      • Versluis M.
      Monodisperse versus polydisperse ultrasound contrast agents: Non-linear response, sensitivity, and deep tissue imaging potential.
      ). In a preliminary in vivo experiment, the measured sensitivity increase was reported to be similar (
      • Jeannot V.
      • Helbert A.
      • Lassus A.
      • Gaud E.
      • Segers T.
      • Botteron C.
      • Frinking P.
      In vivo evaluation of monosize microbubbles: Acoustic efficiency and safety.
      ). The in vivo use of monodisperse bubbles is, however, still at an early stage, owing to the very recent gain of control over the full production process necessary to enable the testing phase. The expected improvement of therapeutic and molecular imaging applications using monodisperse agents also remains to be characterized in vivo.

      Microfluidics to produce complex agents

      The success of microbubbles and ultrasound imaging has led to the creation of the large diversity of contrast agents, as discussed in this review. The same limitations and challenges apply to controlling the response and properties of microfluidic agents to maximize their effect, for both contrast enhancement and for therapy. Here again, microfluidic techniques have the potential to address these requirements and are of special interest for the production of particle-loaded microbubbles, (

      Seo Minseok, Gorelikov Ivan, Williams Ross, Matsuura Naomi. Microfluidic assembly of monodisperse, nanoparticle-incorporated perfluorocarbon microbubbles for medical imaging and therapy. Langmuir 2010;26:13855–13860. ACS Publications.

      ;
      • Peyman S.A.
      • Abou-Saleh R.H.
      • McLaughlan J.R.
      • Ingram N.
      • Johnson B.R.G.
      • Critchley K.
      • Freear S.
      • Evans J.A.
      • Markham A.F.
      • Coletta P.L.
      • Evans S.D.
      Expanding 3 D geometry for enhanced on-chip microbubble production and single step formation of liposome modified microbubbles.
      ), of hard-shelled agents (
      • Lee S.S.
      • Park J.W.
      • Pelet S.
      • Hegemann B.
      • Jeon N.L.
      • Peter M.
      Microfluidic-Based Assay Platform for Studying Polarization Mechanism of Budding Yeast Under Gradient of Mating Pheromone.
      ;
      • Abbaspourrad A.
      • Duncanson W.J.
      • Lebedeva N.
      • Kim S.H.
      • Zhushma A.P.
      • Datta S.S.
      • Dayton P.A.
      • Sheiko S.S.
      • Rubinstein M.
      • Weitz D.A.
      Microfluidic fabrication of stable gas-filled microcapsules for acoustic contrast enhancement.
      ), of multi-layered bubbles (
      • Hettiarachchi K.
      • Lee A.P.
      • Zhang S.
      • Feingold S.
      • Dayton P.A.
      Controllable microfluidic synthesis of multiphase drug-carrying lipospheres for site-targeted therapy.
      ;
      • Shih C.P.
      • Chen H.C.
      • Chen H.K.
      • Chiang M.C.
      • Sytwu H.K.
      • Lin Y.C.
      • Li S.L.
      • Shih Y.F.
      • Liao A.H.
      • Wang C.H.
      Ultrasound-aided microbubbles facilitate the delivery of drugs to the inner ear via the round window membrane.
      ) and of droplet precursors (
      • Hsiung S.K.
      • Chen C.T.
      • Lee G.Bin.
      Micro-droplet formation utilizing microfluidic flow focusing and controllable moving-wall chopping techniques.
      ;
      • Seo M.
      • Matsuura N.
      Mondisperse, submicrometer droplets via condensation of microfluidc-generated gas bubbles.
      ). Although these approaches were met with preliminary success, they often require a modification of the chip and/or of the process driving the chip. Satisfactory microfluidic production of these agents therefore requires revisiting our understanding of bubble production to account for these specificities. Such developments are still in their infancy.

      Scale-up challenge

      A single flow-focusing device with a single nozzle can deliver a clinical dose in seconds to minutes, which potentially allows for the on-demand production of microbubbles at the bedside. However, in an industrial approach, the production rate achievable with a single microfluidic chip remains insufficient. A relevant production rate at a reasonable cost requires the parallelization of tens or hundreds of microfluidic nozzles. Some attempt has been made in this direction, with fewer nozzles that led to either a loss of control or a low production rate (
      • Hashimoto M.
      • Shevkoplyas S.S.
      • Zasońska B.
      • Szymborski T.
      • Garstecki P.
      • Whitesides G.M
      Formation of bubbles and droplets in parallel, coupled flow-focusing geometries.
      ;
      • Mullin L.
      • Molly K.
      • Rothstein Jonathan P
      Scale-up and control of droplet production in coupled microfluidic flow-focusing geometries.
      ;
      • Borden M.A.
      • Streeter J.E.
      • Sirsi S.R.
      • Dayton P.A.
      In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.
      ;
      • Jeong H.H.
      • Yadavali S.
      • Issadore D.
      • Lee D.
      Liter-scale production of uniform gas bubbles via parallelization of flow-focusing generators.
      ), highlighting the underlying difficulties. Cross talk between nozzles remains a question because the fluid dynamics in such a system remain yet to be investigated. Scale-up is currently the main challenge for the industrial translation of microfluidic techniques (
      • Christian H.
      Large-scale droplet production in microfluidic devices - an industrial perspective.
      ). Other techniques that have been explored for microbubble production include electrohydrodynamic atomisation (
      • Farook U.
      • Stride E.
      • Edirisinghe M.J.
      Preparation of suspensions of phospholipid-coated microbubbles by coaxial electrohydrodynamic atomization.
      ), electrolytic techniques and hybrid approaches (
      • Parhizkar M.
      • Stride E.
      • Edirisinghe M.
      Preparation of monodisperse microbubbles using an integrated embedded capillary T-junction with electrohydrodynamic focusing.
      ). All these, however, also suffer from relatively low production rates in their current format.

      Approval of New Products

      As described in the previous section of this report, multiple methods have been investigated to produce microbubbles. Each technique exhibits strengths and weaknesses in terms of ease of use, cost effectiveness, production yield, control of the microbubbles size and polydispersity, stability, energy use, etc. However, translation of these innovative procedures to the clinic and to the market faces numerous hurdles in terms of scalability, translatability and regulation. In fact, in contrast to conventional small pharmaceutical drugs, microbubble constructs are rather complex with various ingredients, typically including gas, lipids, excipients and targeting ligands, making the pharmaceutical development quite challenging. Here, we will outline a possible path to be followed for a successful translation of novel agents, focusing on lipid-shelled gas-filled microbubbles that represent the majority of commercially available ultrasound contrast agents. It should be emphasised that, although they are used for diagnostic purposes, gas microbubbles are currently considered as a medicinal drug and thus must follow the same development path as therapeutic drugs. Their designation for therapeutic applications remains to be determined.

      Chemistry, manufacturing and controls

      Chemistry, manufacturing and controls (CMC) and regulatory affairs (RA) are of paramount importance at the various stages of pharmaceutical product development. Regulatory agencies, such as the United States Food and Drug Administration (FDA), European Medicines Agency and UK Medicines and Healthcare Products Regulatory Agency (MHRA) provide some guidance and recommendations on the CMC activities to be carried out to guarantee safe, effective and high-quality products (
      United States Food and Drug Administration
      Bioanalytical method validation guidance for industry biopharmaceutics bioanalytical method validation guidance for industry biopharmaceutics contains nonbinding recommendations.
      ). The CMC development plan typically involves the following three main parts that an applicant must submit as part of the quality dossier for any new drug application, Investigational Medicinal Product Dossier or investigational new drug filings: (i) characterization of the manufacturing components and materials, (ii) product manufacturing procedures and (iii) product testing. These CMC activities are involved at the various stages of the microbubble pharmaceutical life cycle.

      Characterization of the manufacturing components and materials

      Microbubbles are generally available as a liquid suspension or as a freeze-dried powder for reconstitution. The applicant should provide information related to the microbubble components such as gas core, lipid ingredients, excipients and targeting ligand moiety in the case of ultrasound molecular imaging applications. The content (mg/vial or mg/g) of each component of the microbubble formulation should be clearly defined and their ranges indicated based on results gathered during the product development stages. Data demonstrating the quality and safety of each ingredient are compulsory. In particular, the applicant should provide in-depth characterization of the microbubble main components such as core gas and lipids (specifications, stability data and microbiologic testing, etc.), quality data have to be similar to those for the submission of drug substance (
      International Council for Harmonisation
      ICH guideline Q11 on development and manufacture of drug substances (chemical entities and biotechnological/biological entities).
      ). In this regard, to ease the regulatory path, the selection of the pharmaceutical gas and lipid components is key for the development of new agents.

      Product testing

      Physicochemical properties of the gas-filled microbubbles are crucial for quality, efficacy and safety considerations. The applicant should develop and validate dedicated analytical procedures (non-GMP and GMP), in line with current regulations and guidelines, for the comprehensive characterization of both drug substance (gas, phospholipids, etc.) and microbubble drug product, and execution thereof according to the CMC plan. Moreover, the applicant must design stability studies to determine the shelf life according to International Council for Harmonisation (ICH) guidelines (
      International Council for Harmonisation
      ICH Topic Q 1 A (R2) stability testing of new drug substances and products step 5 note for guidance on stability testing: Stability testing of new drug substances and products.
      ) with in-depth characterization of impurities and possible degradation products. In fact, stress and accelerated stability testing data are mandatory to determine the degradation profile, establish stability indicating analytic methods, define appropriate storage conditions ensuring the microbubble quality and performances and ultimately define retest periods. In addition to safety and quality considerations, microbubble shelf life and stability of the reconstituted suspension are important elements from the practical perspective.
      The following properties are generally monitored: microbubble size (i.e., mean and distribution profile) and concentration, surface charge or zeta potential, osmolality, viscosity and resistance to hydrostatic pressure, shell lamellarity and mechanical properties. Moreover, residual solvents must be monitored if any organic solvent is used during the manufacturing process. Lipid and gas contents are also systematically determined using dedicated analytic procedures, namely liquid chromatography and gas chromatography. Because microbubbles are sterile products, microbiology activities, such as sterilization validations, endotoxin testing, sterility testing and container closure testing and other USP microbiology tests, should be performed as recommended by regulatory authorities. Critical quality attributes (CQAs) of the contrast agent can thus be defined.
      In vitro measurements of the agent's acoustic properties, such as attenuation and backscatter coefficients as a function of frequency, are also important features to quantify. In addition, mechanical properties (i.e., shell stiffness) are also determined using appropriate methods, e.g., backscatter and attenuation (
      • Gorce J.M.
      • Arditi M.
      • Schneider M.
      Influence of bubble size distribution on the echogenicity of ultrasound contrast agents.
      ), high-speed optical imaging (
      • van der Meer S.
      • Dollet B.
      • Voormolen M.
      • Chin C.T.
      • Bouakaz A.
      • de Jong N.
      • Versluis M.
      • Lohse D.
      Microbubble spectroscopy of ultrasound contrast agents.
      ) and atomic force microscopy (

      Sboros V, Mcdicken WN, Koutsos V. Nanomechanical probing of microbubbles using the atomic force microscope. 2007;46:349–354.

      ). Finally, in vitro stability of the agent after incubation in plasma at 37°C in a time window compatible with clinical examination can also be relevant information for stability assessment.

      Manufacturing process

      According to the ICH guideline for industry (Q8(R2) Pharmaceutical development), the manufacturing process has to be fully described with a detailed flow diagram (batch size, bulk and finished drug product, purification and sterilization methods, freeze-drying, packaging, etc.) and validated to demonstrate the consistent production of a safe and effective agent in line with the specifications set in the filling dossier (new drug application). Microbubble manufacturing under cGMP conditions for phases I, II and III clinical trials includes acquisition and identification testing of incoming materials (gas, lipids, excipients or non-active ingredients, etc.), drafting the master batch record, compounding, filling, labeling and release testing.
      All through the process scale-up to generate larger batches for commercialization purposes, the process development should yield a product satisfying the CQAs identified during formulation development activities and clinical trials. To mitigate the risk and ensure the microbubble performances in terms of safety and efficacy, it is highly recommended to use quality by design chemometric approach (in contrast to conventional empirical methodology) and to gain better knowledge on the correlation between critical material attributes, critical process parameters and the product CQAs. Establishing an extended design space will enable more flexible regulatory approaches (
      • Yu L.X.
      Pharmaceutical quality by design: Product and process development, understanding, and control.
      ).
      Although not new, data integrity is gaining increased attention at different levels of the pharmaceutical product development. In fact, regulatory bodies such FDA and MHRA have released new cGMP guidelines emphasizing the central role of data integrity to ensure that the end-product meets the required quality standards over its entire life cycle. In this respect, computerized systems must be validated to guarantee data accuracy, completeness and consistency. Therefore, pharma professionals including manufacturing and quality management are asked to make certain seamless documentation of all information pertaining to products and processes from collection and storage of the data to its destruction.

      Pharmacodynamics, Pharmacokinetics and toxicology

      Regulatory approval of new drugs commonly involves preclinical, clinical and post-marketing phases. The main objective of preclinical study activities is to assess the pharmacokinetic and safety profile and the pharmacodynamics of the contrast agent. To anticipate clinical trials in human patients and ensure patient safety, in vitro and in vivo, experiments are performed in compliance with good laboratory practice (GLP) regulations to determine the maximum tolerated dose of a diagnostic agent and identify possible adverse effects. At this stage, the appropriate selection of relevant animal models is fundamental to ensure a seamless transition to the clinic. The maximum tolerated dose is determined through a dose escalation approach. To demonstrate the effectiveness of the enhancing agent, a diagnostic index can be used to describe the ratio of the dose causing toxicity and the dose eliciting a signal enhancement.
      Pharmacokinetic studies are meaningful to establish dosing regimens and develop dose-concentration versus response relationships. After intravenous injection of the agent at various doses, the pharmacokinetic profile is acquired to assess dose dependency, plasma clearance and elimination pathway. Pharmacokinetics and bioavailability for microbubble studies are also performed within a GLP environment, using validated bioanalytical procedures (
      United States Food and Drug Administration
      Bioanalytical method validation guidance for industry biopharmaceutics bioanalytical method validation guidance for industry biopharmaceutics contains nonbinding recommendations.
      ;
      European Medicines Agency
      Committee for Medicinal Products for Human Use (CHMP) guideline on bioanalytical method validation.
      ). The blood kinetic and elimination of the gas can be accomplished by gas monitoring in blood and exhaled air analysis, using highly sensitive gas chromatography mass spectrometry methods (
      • 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.
      ;
      • Li P.
      • Hoppmann S.
      • Du P.
      • Li H.
      • Evans P.M.
      • Moestue S.A.
      • Yu W.
      • Dong F.
      • Liu H.
      • Liu L.
      Pharmacokinetics of perfluorobutane after intra-venous bolus injection of sonazoid in healthy Chinese volunteers.
      ). In parallel, by radiolabeling key moieties of the microbubbles (e.g., phospholipid), a mass balance can be achieved through the collection and testing of blood, urine and fecal samples.
      Preclinical in vivo toxicology studies, beginning with a single dose followed by a repeated doses approach, enables the identification of a suitable and safe beginning dose for clinical trials. Other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity are carried out during the preclinical phase of development, depending on the application purpose. Based on the preclinical studies outcome, clinical phases I, II and III are conducted to further demonstrate the safety and efficacy of the imaging agent in healthy patients and patients who are ill.

      Conclusion

      In addition to exciting developments in targeted imaging in the diagnostic field, there is a broad wave of advances in applying microbubbles to various therapeutic applications, notably in the delivery of drugs across the blood–brain barrier and in the treatment of challenging solid tumours. Other new agents are currently under development such as sub-micrometer liquid droplets, gas entrapping nanoparticles, monodisperse agents and phase-change contrast agents with promising therapeutic applications. Bringing these new agents from bench to bedside requires some specific expertise in terms of large-scale cGMP manufacturing, pharmaceutical design, quality and risk management, regulation and preclinical and clinical assessment. The modern pharmaceutical market is however characterized by an increased drug development cost, shorter product life cycles and global competition. A new development pathway is therefore a necessity, and close collaboration between ultrasound and pharmaceutical companies is essential. Finally, close interaction with regulatory bodies is key to ensure a seamless translation of these new agents from laboratory bench to patient bedside.

      Acknowledgments

      The authors gratefully acknowledge funding from the UK Engineering and Physical Sciences Research Council (grants EP/I021795 and EP/LO24012 ) and US National Institutes of Health (grant R01 CA195051 ).

      Declaration of Competing Interest

      TB and SC are full time employees of Bracco Suisse. The other authors are hold full time academic positions and have the following company affiliations: ES is a founder and non-executive director of SonoTarg Ltd.; MB is a founder and chief scientific officer of Respirogen Inc.

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