Applications of sub-micron low-boiling point phase change contrast agents for ultrasound imaging and therapy

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Abstract

Phase change contrast agents (PCCAs) have been studied in the medical ultrasound field for nearly three decades. Their ability to convert from a liquid core droplet to an acoustically active microbubble has enhanced the possibilities of medical ultrasound, enabling new imaging approaches as well as therapeutic directions. However, traditional PCCAs are formulated with perfluorocarbons which are a liquid at standard temperature and pressure, requiring a high amount of energy to transition the encapsulated droplets to gas form, possibly resulting in undesired bioeffects. A new generation of low-boiling point PCCAs, which are formulated from gaseous perfluorocarbons in a metastable liquid state, seeks to overcome these limits. These super-heated liquid perfluorocarbon nanodroplets display longer circulation kinetics than microbubbles, their activation produces unique acoustic signatures, and their small particle size holds potential for extravascular applications. Low-boiling point nanodroplets can be phase-transitioned when activated with ultrasound at pressures and frequencies approved for diagnostic imaging. From the first publication almost 10 years ago, low-boiling point PCCA research has expanded rapidly, and recent advances in super-resolution imaging, drug delivery and neuromodulation made possible by these nanodroplets are just a few examples of this growing field of research. In this review, we discuss low-boiling point phase change contrast agents and their applications in ultrasound imaging and therapeutics.

Graphical abstract

A. Depiction of the neuromodulatory potential of low-boiling point PCCAs in anesthetizing local brain regions by focused delivery of anesthetics.. B. Clot disruption with ultrasound (sonothrombolysis) is greatly enhanced by the unique erosion pattern observed when combined with low-boiling point PCCAs.

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Introduction

In the late 1960s, Gramiak et al. observed the acoustic signature of agitated saline microbubbles while imaging an aortic root, sparking the inception of ultrasound contrast agents [1]. The first commercial stabilized microbubble contrast agent using albumin was developed in the late 1980s, with the commercialization of Albunex (Molecular Biosystems Inc.). The introduction of microbubble contrast agents expanded the possibilities of acoustic imaging and therapy, ushering in a new era that now includes several commercial manufacturers globally and many more variants in development [2].

Approximately 30 years following the approval of clinical microbubbles, the “condensed” microbubble was demonstrated by Sheeran et al. [3,4]. Approximately five times smaller in diameter than the original micron-sized precursor bubbles, these liquid-core particles (now commonly referred to as nanodroplets or phase change nanoemulsions) were produced by condensing their gaseous core into a liquid through increased pressure and decreased temperature. The resulting liquid-core droplets could be vaporized upon ultrasound application, allowing these phase-change contrast agents (PCCA) to be activated, returning to microbubble form on demand. This ultrasound-mediated phase change of the liquid core to the gas phase is referred to as acoustic droplet vaporization (ADV).

Prior to condensed microbubbles, phase change contrast agents had been studied since the late turn of the century, described by Correas et al. and Apfel et al. in the late 1990s [5,6] and Kripfgans et al. in the early 2000s [7,8]. This first generation of PCCAs were directly formulated as an emulsion from liquid perfluoropentane with diameters of a few microns, but could be vaporized with high pressure. The energy required for ADV is dependent on the boiling point of the perfluorocarbon, droplet size, and the properties of the shell material. Once vaporized, the generated microbubble can be effectively used in either imaging or therapeutic applications with ultrasound. Traditional PCCAs have demonstrated many applications, such as aberration correction [9], embolotherapy [10,11], cavitation enhancement [12, 13, 14], therapeutic delivery [15,16], and contrast agents both for ultrasound and photoacoustics [17, 18, 19].

In comparison to microbubbles or traditional PCCAs, condensed microbubbles (nanodroplets in this article), have a liquid core of a perfluorocarbon which is a gas at room temperature and pressure under standard laboratory conditions. There are two perfluorocarbons with low-boiling points, decafluorobutane (DFB, b.p. −2 °C) and octafluoropropane (OFP, b.p. −37 °C), which to date have been used in these contrast agents, which provides the term for these condensed microbubbles as “low-boiling point PCCAs”. These nanodroplets can be formulated not only from precursor bubbles, but also through other techniques to produce a stable size distribution typically on the order of 100–300 nm in diameter. Although technically, their diameter (>100 nm) is too large to be considered “nanoparticles” in FDA terms, the term nano is often still used to differentiate these PCCAs from microbubbles which otherwise average around 1 micron in diameter.

Contrary to what one might expect considering the perfluorocarbons utilized for their cores, which would normally be a gas below the freezing point of water, these nanodroplets remain metastable in liquid form even at temperatures well above the bulk boiling point of the perfluorocarbon core, due to homogenous nucleation and perfluorocarbon intermolecular forces [20]. As a result, these low-boiling point PCCAs can undergo ADV at much lower acoustic pressures than those required to vaporize traditional PCCAs (i.e., perfluoropentane), enabling their use in a wider variety of biomedical applications where minimizing high acoustic energies is desirable to avoid bioeffects. In short, a low-boiling point nanodroplet PCCA can be converted into a traditional “microbubble” contrast agent with diagnostic imaging parameters. As with the initial development of the stabilized microbubble, the low-boiling point perfluorocarbon nanodroplet has welcomed a second wave of possibilities for ultrasound imaging and ultrasound-mediated therapy.

After ADV, low-boiling point PCCAs are converted to microbubbles and can be used as contrast agents. Microbubble density and compressibility differs substantially from blood and other physiologic tissues. The resulting mismatch in acoustic impedance turns a microbubble into a scatterer which is easily detectable with ultrasound. Beyond the greatly increased acoustic scatter, micron-scale bubbles have a unique acoustic signature, oscillating in an acoustic field due to their compressible nature, producing harmonics and subharmonics. This oscillation can be used to isolate the bubble signal from tissue to define vascular structures and is the basis of ultrasound contrast imaging. At higher acoustic pressures, microbubbles can be driven to collapse and fragmentation. These effects can be leveraged for therapeutic applications to increase physical disruption or tissue heating with ultrasound. However, prior to ADV, low-boiling point PCCAs demonstrate substantial advantages over microbubbles. With diameters on the order of a few hundred nanometers, they have an increased potential to leave the vasculature, whereas microbubbles are generally confined to blood vessels due to their micron size. As the core is liquid instead of gas, PCCAs have a circulation half-life that is longer than a microbubble of the same perfluorocarbon due to decreased dissolution [21,22]. Furthermore, PCCAs are poorly acoustically active prior to ADV. This can provide a significant advantage over microbubbles for selectively enhancing or affecting a localized region of interest, since image contrast or bioeffects will be generated only at locations where ADV has been initiated. These benefits, along with unique ADV-related acoustic emissions that provide a means to isolate the echo signature of PCCAs from bubbles and tissue, open the door to unique applications for nanodroplets that are beyond what is currently achievable with microbubbles. This review highlights recent applications of low-boiling point nanodroplets composed of octafluoropropane (OFP) or decafluorobutane (DFB) in imaging and therapy.

Section snippets

Recent advances in droplet manufacturing

Liquid perfluorocarbon emulsions have been investigated as intravenous gas carriers for decades, due to their ability to solubilize oxygen, carbon dioxide, and other blood gases at high capacity [23,24]. Much early work used long-chain perfluorocarbons, such as perfluorooctyl bromide, which are liquid at room temperature and can be emulsified using traditional formulation strategies. These perfluorocarbons have high-boiling points, affording facile emulsion preparation but requiring

Recent applications for imaging

Low-boiling point PCCAs have a unique advantage in imaging applications. Their long circulation lifetime can enable extended contrast life with a single bolus [35], they can be vaporized into bubbles on demand, and their activation produces a unique activation signature [36]. Early droplet imaging work simply took advantage of improved circulation kinetics, such as enhanced transthoracic myocardial contrast enhancement in pigs following IV delivery [37]. This increased signal allowed imaging of

Recent applications for therapy

Since the introduction of contrast for imaging applications, researchers and regulatory agencies have sought to understand the biological effects of combining microbubbles and ultrasound. These investigations have revealed that microbubble contrast can be administered and imaged safely but have also elucidated many mechanisms whereby microbubbles can cause biological effects. Some of these mechanisms, while undesired in imaging applications, may be exploited for therapeutic applications.

Other applications

In addition to therapeutic and imaging applications, nanodroplets are finding uses in diagnostic areas both in vitro and in vivo. As ultrasound is used widely across many industries, and at large and small scales, it is possible for nanodroplets to have manufacturing or other non-biomedical uses. Described in this section are recent developments in droplet applications that deviate from traditional uses of nanodroplets as an improved microbubble.

Safety

The potential of microbubbles to cause bioeffects can raise safety concerns for diagnostic imaging, but can also make microbubbles useful therapeutic tools. Decades ago, diagnostic ultrasound faced a similar challenge, and eventually, standards on ultrasound exposure for clinical imaging were established that were recognized as safe. One such standard is the mechanical index (MI), a metric proportional to ultrasound pressure and frequency and which indicates the likelihood of cavitation

Conclusions

Low-boiling point PCCAs offer many of the benefits of traditional microbubbles or traditional PCCAs for imaging and therapy, yet with several notable advantages. Nanodroplets provide enhanced circulation kinetics, small particle size, and capacity for extravasation or penetration beyond the reach of microbubbles. Unique acoustic responses, such as harmonic emissions and stochastic activation patterns, are unique to phase-change particles and are being explored to improve contrast imaging

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P.A.D. is a co-inventor on issued and pending patents describing phase change contrast agent technologies as well as ultrasound imaging technologies. He is also a co-founder of Triangle Biotechnology Inc. and SonoVol Inc., both of which have licensed patents of which he is a co-inventor. P.A.D. and P.G.D. are co-inventors on a pending patent describing biofilm

Acknowledgements

The authors appreciate editorial assistance from Virginie Papadopoulou, Isabel Newsome, and James Tsuruta. Research on phase change contrast agents in the Dayton Laboratory has been supported in part by the National Institutes of Health grants R21EB021012, R33CA206939, R01HL141967-01A1, R01CA220681, R01CA232148, R43CA236177, and R21EB021103-01A1, as well as pilot funding from the Carolina Center for Cancer Nanotechnology Excellence. The financial sponsors had no role in the content of this

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      Citation Excerpt :

      Furthermore, their selective activation potential, resulting in phase conversion to an acoustically active state only after sufficient energy deposition, makes them useful for high-intensity focused ultrasound (HIFU) and local targeting applications (Moyer et al. 2015). Because of these advantages, PCCAs are a growing research interest for ultrasound therapy and imaging applications, many of which have been reviewed recently (Lea-Banks et al. 2019; Borden et al. 2020; Durham and Dayton 2021). Phase-change contrast agents consist of a perfluorocarbon core surrounded by a shell material.

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