Original ContributionInvestigation of the Acoustic Vaporization Threshold of Lipid-Coated Perfluorobutane Nanodroplets Using Both High-Speed Optical Imaging and Acoustic Methods
Introduction
Gas-filled microbubbles, stabilized by a coating material such as phospholipids, denatured human serum albumin or synthetic polymers, have been the subject of extensive investigation both as ultrasound contrast agents and as therapeutic carriers, for example, for drug delivery and gene therapy (Liu et al. 2006; Hernot and Klibanov 2008). Their size (1–10 µm), however, limits both their circulation time and their ability to extravasate and accumulate in a target tissue (Kaya et al. 2010). Lipid-coated perfluorocarbon (PFC) “nano”droplets (NDs) with diameters of a few hundred nanometres have been explored as a means of addressing these limitations (Zhou et al. 2013). Please note that the NDs described here do not meet the strict definition of “nano,” that is, smaller than 100 nm, but the term has become widely used in the literature. The lipid shell coating the PFC core can help to stabilize the NDs and facilitates biocompatibility and functionalization of the ND surface to enable targeting and/or attachment of therapeutic species (Unger et al. 2004; Hatziantonioy and Demetzos 2008; Peetla et al. 2013). PFC NDs are not easily detected by ultrasound imaging because of their liquid core and size. Upon exposure to ultrasound of sufficient intensity, however, they can be converted into echogenic gas-filled microbubbles through a process termed acoustic droplet vaporization (ADV) (Kripfgans et al. 2000; Matsuura et al. 2009; Sheeran et al. 2011c). Because of the high Laplace pressure and corresponding increase in the energy required to vaporize the encapsulated liquid, the acoustic pressures typically required for ADV are much higher than those required for stimulating microbubbles (Mannaris et al. 2019). This can increase the probability of unwanted bio-effects (Dalecki 2004; Leighton 2012), and consequently, a range of different methods have been explored for reducing the pressure threshold for ADV.
Perfluoropentane (PFP) and perfluorohexane (PFH) have been most commonly used to form the core of NDs, but these both require substantial acoustic pressures to achieve vaporization (Kripfgans et al. 2000; Fabiilli et al. 2009; Matsuura et al. 2009; Zhang and Porter 2009; Vlaisavljevich et al. 2015a, 2015b). Even for therapeutic applications, in which higher ultrasound intensities are normally used, vaporization efficiency may be poor and recondensation of droplets can occur after vaporization (Reznik et al. 2013; Shpak et al. 2014). One approach to solve this has been to use a mixture of droplets and microbubbles. The inertial collapse of the microbubbles at moderate ultrasound intensities is thought to trigger ADV through the localized generation of shock waves (Lo et al. 2007; Healey et al. 2016a). “Acoustic cluster therapy” (ACT) is an example of this approach, although currently the size of the clusters used limits its application to targets where vascular embolization is desirable (Sontum et al. 2015; Healey et al. 2016b; Wamel et al. 2016). Incorporation of solid nanoparticles to act as nuclei within the droplets has also been used to successfully lower the ADV threshold of NDs (Lee et al. 2015), but it is not always desirable to include additional materials in the formulation, and tolerability concerns over the biomedical use of nanoparticles remain. Using alternative PFCs with lower boiling points is another way to reduce the ADV threshold (Sheeran et al. 2011a, 2011b, 2011c; Rojas et al. 2019). Sheeran et al. (2011a, 2011b) proposed a method whereby perfluorobutane (PFB) and octafluoropropane (OFP), which are gaseous at room temperature, can be used to produce both nano- and microdroplets (MDs, i.e., >1 µm in diameter) by a microbubble condensation technique (Sheeran et al. 2011a, 2012). They found that NDs/MDs produced in this way required significantly lower pressures for ADV compared with similar droplets of PFP or PFH.
In addition to the droplet composition, it has been found that many other parameters influence the ADV threshold of PFC ND/MDs. These include environmental parameters (such as temperature, viscosity of the surrounding fluid and boundary conditions); droplet characteristics (size and concentration, as well as core and shell composition); and the acoustic exposure parameters (frequency, pulse repetition frequency, pulse length and exposure duration). Perhaps as a consequence of this sensitivity to multiple parameters, there is considerable variation in the published values for ADV thresholds in the literature, as outlined in Table 1, which summarises the results from 29 studies of PFC ND/MD vaporization. There are some qualitatively consistent trends. For example, the ADV threshold typically decreases with increasing environmental temperature, tube diameter, droplet size and concentration, pulse repetition frequency and pulse length (Kripfgans et al. 2000; Lo et al. 2007; Porter and Zhang 2008; Fabiilli et al. 2009; Aliabouzar et al. 2018; Rojas et al. 2019). There are, however, differences across studies concerning the effect of ultrasound frequency. In some studies, the ADV threshold increases with increasing ultrasound frequency (Kripfgans et al. 2004; Sheeran et al. 2013b; Vlaisavljevich et al. 2015a; Aliabouzar et al. 2018), which is consistent with classic nucleation theory (Vlaisavljevich et al. 2016). However, an opposite effect has also been reported (Kripfgans et al. 2000, 2002; Schad and Hynynen 2010a; Williams et al. 2013). These contradictory results have been attributed variously to limitations of the experimental apparatus, droplet deformation (Kripfgans et al. 2004) and, in the case of MDs, non-linear propagation and superharmonic focusing (Shpak et al. 2014; Miles et al. 2016).
A further confounding factor is the fact that the definition of the threshold itself may vary between studies and according to the measurement technique(s) used. Both direct and indirect methods have been applied. Direct measurements include high-magnification microscopy and high-speed imaging, enabling direct observation of the vaporization process (Kripfgans et al. 2004; Sheeran et al. 2013b; Vlaisavljevich et al. 2015a). However, optical observation is not suitable for measuring the initial size of droplets below 800 nm because of the resolution limits of brightfield imaging, nor can it be applied in tissue. To address this, indirect methods, such as ultrasound imaging (Kripfgans et al. 2000; Lo et al. 2007; Porter and Zhang 2008; Fabiilli et al. 2009) and/or monitoring of acoustic emissions (Vlaisavljevich et al. 2015a; Aliabouzar et al. 2018) have been used to identify ADV. In all cases the sensitivity and/or spatial resolution of the system will affect the pressure at which a bubble (or bubbles) or its emissions are first detected and, hence, the recorded threshold. A further important distinction with acoustic methods is whether it is the first appearance of a gas bubble(s) that is being detected, that is, true ADV, or its subsequent oscillation and collapse. Under the acoustic exposure conditions typically required for ADV the resulting bubble will be likely to undergo inertial cavitation (IC), that is, when a bubble grows to a diameter that is at least twice its original diameter during a single cycle of acoustic pressure and then collapses violently under the inertia of the surrounding fluid, potentially fragmenting into many smaller bubbles (Neppiras 1980; Fabiilli et al. 2009). The measured threshold, however, will depend upon the signal amplitude selected by the experimenter as representing ADV or IC. This is discussed further, later.
The thresholds determined by different methods may also vary on account of the stochastic nature of both ADV and IC. If a droplet of a given size has a fixed probability of vaporising at a given ultrasound frequency and pressure, then the larger the number of droplets present, the more likely it is that an ADV event will occur. The same applies to bubbles and IC. The field of view in an optical experiment will typically be considerably smaller than that of an ultrasound transducer and so contain fewer ND/bubbles. This can potentially lead to a higher threshold being measured by optical compared with acoustic methods. In addition, there will also likely be a range of ND/bubble sizes present, the probability of ADV/IC may vary with other parameters, for example, differences in coating integrity; and, once some bubbles have formed, their collapse may promote ADV as mentioned above.
Despite the desirability of using PFB or OFP to minimize the ADV threshold, there have been relatively few studies that systematically investigate their vaporization dynamics. Sheeran et al. (2011c) investigated the effect of Laplace pressure on the vaporization threshold of different PFC MDs (1–13 μm), and found the vaporization thresholds of PFB MDs were lower than thresholds of the higher-boiling point PFC MDs and decreased as the MD diameter increased. More recent studies by Sheeran et al. (2013b) found that the vaporization threshold for PFB NDs increased with ultrasound frequency. These findings are further supported by Rojas et al. (2019), who investigated the effect of environmental parameters (including hydrostatic pressure, boundary constraints and viscosity) on the vaporization threshold of PFB NDs. There remains, however, considerable uncertainty regarding the activation and subsequent dynamics of low-boiling-point PFC NDs. The aim of this study is therefore to undertake a comprehensive investigation of both the ADV and IC thresholds of lipid-coated PFB NDs using a combination of high-speed video microscopy, B-mode ultrasound imaging and passive cavitation detection. The effects of acoustic parameters (pulse repetition frequency, pulse length and frequency), in addition to droplet parameters (droplet composition, size and concentration) and temperature, on the vaporization threshold of PFB NDs are all investigated.
Section snippets
Materials
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol, glycerol, propylene glycol and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (Gillingham, Dorset, UK). PFB and PFP were obtained from FluoroMed, L.P. (Round Rock, TX, USA). PFB was chosen in preference to OFP for this study on the basis of
ND size and concentration
For the PFB NDs used in the majority of the experiments, the mean diameter measured over five different batches with DLS was 237 ± 16 nm (mean ± standard deviation), as illustrated in Figure 5b. The corresponding concentration, as measured by NTA, was 6.6 ± 0.4 ×1011 droplets/mL. For all experiments except those in which concentration was a variable, the suspension was diluted by a factor of 100. For the experiment in which size and composition were varied, both PFB and PFP NDs were prepared
Effect of PRF and pulse length
Both the ADV and IC thresholds decreased in a similar fashion with increasing PRF and increasing pulse length (Figs. 9 and 10). This is consistent with studies of PFP NDs (Lo et al. 2007; Fabiilli et al. 2009) and is likely associated with increasing probability of ADV or IC. If the probability of ADV or IC for a single ND or bubble has a fixed value, then increasing either the PRF or pulse length would increase the expected number of events over the course of the experiment.
Effect of driving frequency
As discussed in the
Conclusions
The aim of this study was to investigate the vaporization of low-boiling-point (PFB) NDs using both optical and acoustic methods over a range of therapeutically relevant exposure conditions. The results complement those of previous studies, as outlined in Table 1, by extending the range of parameters investigated, thus enabling a more comprehensive understanding of the behavior of these agents. To the best of the authors’ knowledge, this is also the first reported high-speed-camera (>1 Mfps)
Acknowledgments
The authors are grateful to the Engineering and Physical Sciences Research Council (EPSRC) for supporting this research through Grant EP/R013624/1. The authors also thank James Fisk and David Salisbury for construction of the apparatus used in this study.
Conflict of interest statement
The authors declare no conflicts of interest.
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