Cavitation microstreaming and stress fields created by microbubbles
Introduction
The therapeutic use of microbubble-mediated ultrasound in medicine is being increasingly explored for mainstream clinical adoption. Many reports, both clinical and in vitro, have been made in the biomedical literature on the microbubble-enhanced sonothrombolytic effect [1], [2], [3], [4], drug or oxygen delivery [5], [6], and sonoporation [7], [8], [9], [10], [11], [12], [13], [14]. Recently, microbubbles and focused ultrasound have been used to transfer large molecules, including an antibody to the Alzheimer’s disease peptide Aβ, across the blood–brain barrier (BBB) [15], [16]. Early speculation on the mechanisms behind such observations focused on the cavitation created at high ultrasound power, in which cell membranes are penetrated in a relatively violent process that can damage tissue [13], [14]. An alternative, gentler phenomenon known as cavitation microstreaming has more recently been proposed as the mechanism driving effective sonoporation and sonothrombolysis [4], [8], [9], [10], [17]. Laboratory studies of microstreaming date back to the 1950s [18], [19]. The first studies of microstreaming by Elder [19] involved elucidating microstreaming around a bubble on a wall at various oscillation amplitudes and fluid viscosities. Though cavitation microstreaming is gentler than cavitation, microstreaming forces can still be intense enough to lyse an artificial vesicle [20].
Like macroscopic acoustic streaming, microstreaming is a second-order nonlinear effect in which velocity gradients in the first-order oscillatory sound field allow rectification of the oscillation, generating a mean flow. Microstreaming relies on the large velocity gradient due to small length scales of the bubble. Of the known theoretical studies that have been made of microstreaming (e.g. [21], [22], [23]), most are analyses of bubbles in an unbounded domain. In cases where the effect of a boundary on microstreaming has been considered, theory has allowed the magnitude of the microstreaming velocity to be computed, but has not contributed insight into the patterns associated with the microstreaming [23]. Microstreaming in the vicinity of boundaries is particularly important, given the possibility that the gentler streaming action on the walls of the vasculature could be relevant for therapeutic purposes [24] (in contrast to the first-order inertial cavitation flow, which may damage cells).
Recent studies into BBB disruption – desirable to enable treatment of conditions such as Alzheimer’s disease with large molecules – have affirmed that sonoporation of the BBB can be achieved without violent inertial cavitation occurring [8], [9], [10], [25]. This important observation could be explained by multiple alternative theories on the mechanism by which large molecules pass through the BBB. Two theories are based on molecular passage across the BBB through stretch-activated channels – pores in cell membranes that open based on their sensitivity to changes in mechanical stress [25]. One variant of the theory involving passage through stretch-activated channels is based on the first-order oscillations of contrast-agent bubbles directly causing activation through “cellular massage” (oscillation of the cell membrane) [11], [26]. Studies using the patch-clamp technique showing stretch-activation through direct mechanical stress [26], [27], [28], [29] lend some support to this theory. The other variant of the theory is that the second-order microstreaming flow generates flow fields that develop shear stresses over cell membranes, resulting in tension and stretching on the membrane walls that cause channel activation [8], [9], [10], [25]. In the wider context of studies using endothelial cells, changes or disruptions in typical cell behaviour with the application of shear stresses have been demonstrated [30], [31], [32]. A third theory on molecular passage across the BBB does not involve passage across stretch-activated channels into individual cells, but rather is based on the opening of gaps between otherwise tightly-packed endothelial cells of the BBB. DePaola et al. [32] found that cell–cell gap communication was only disrupted when a steep gradient in the shear stress was present on vascular walls; when a high uniform shear stress was applied, cell–cell gap communication was unaffected. Given that none of these theories has been fully validated for the explanation of sonoporation-enhanced large molecule passage across the BBB, we believe additional metrics need to be introduced, beyond the usual presentation of shear stress numbers in literature with biomedical applications e.g. [7], [23]. Metrics directly linking the characteristics of externally-applied ultrasound to sonoporation outcomes are not feasible in the near future, but indirect linkage through knowledge of induced microstreaming patterns is increasingly feasible.
The present paper has two parts. In the first part, the results database from the experiments of Tho et al. [33] are combined with new data and re-analyzed, with focus on interpretation in the context of possible sonoporative and sonothrombolytic mechanisms due to microbubble microstreaming. Emphasis is placed on the different microstreaming patterns developed. In addition, a new metric – the surface divergence on the cell membrane surface – is introduced as a possible measure of the ability of a cavitation microstreaming flow to attain sonoporation. In the second part, we consider micromixing due to microbubble microstreaming. Effective sonoporation-facilitated transport of drug molecules across the BBB requires that the molecules penetrate down to the tissue surface, such that they are readily available for passage through pores and gaps when they are opened by the microstreaming. Similarly, in the sonothromobolysis of blood clots for the emergency treatment of stroke using an injected drug such as tissue plasminogen activase (tPA) [4], the tPA molecules must also penetrate to the surfaces of the clot for the treatment to be effective. Both applications therefore require effective mixing of therapeutic molecules at the microscale, particularly if it is desirable for systemic high-concentration dosages to be avoided. Over the micron scales typical of individual cells, fluid flows are generally laminar: mixing at such length scales becomes fundamentally problematic [34], [35] and it is thought that microstreaming-induced micromixing may assist therapeutic molecule transport.
Section snippets
Microscope and micro-PIV system
The microscopic particle–image velocimetry (PIV) system (Fig. 1) consists of an inverted epi-fluorescent microscope (Nikon TE2000-E), a 532 nm light source, and a CCD camera imaging system (PCO Sensicam QE). Micro-PIV as a flow measurement technique has been well documented [33], [36], and the details of the current system have been described in detail in [36].
In the present experiments, a continuous 100 W super high-pressure mercury-arc lamp was used as the light source; the light was filtered
Acoustic microstreaming patterns
A number of factors make the present results only illustrative, not definitive, of the phenomena that may be present in real clinical applications. Firstly, owing to the need to measure flow fields with adequate resolution, the present results are for bubbles two orders of magnitude larger than the contrast-agent sized bubbles most likely to be employed in sonothrombolysis, which for intravenous treatment are constrained by the diameter of the pulmonary capillaries to be a few microns in size.
Micromixing
The use of acoustic microstreaming to achieve micromixing within specially-designed devices has been demonstrated by Liu et al. [44]. The current infeasibility of such devices at the micron scale in the cerebral vasculature, and the absence of turbulence, means effective mixing to achieve drug molecule penetration down to cell walls requires alternating flow patterns. Tho et al. [33] showed that acoustic microstreaming can also serve this purpose, because a variety of flow patterns are produced
Discussion and conclusions
It must be emphasised that the present results are for microbubbles much larger that those used in clinical practice. In particular, although microstreaming has been observed around contrast bubbles [17], it is a nonlinear phenomenon and the patterns may not scale; the boundary conditions would be different, and moreover the cellular surface is not rigid. Data on streaming patterns around contrast agents on surfaces, although inevitably of much lower resolution, should be sought in the future.
Acknowledgements
We are grateful to Guy Metcalfe and Daniel Lester at CSIRO for guidance in mixing theory, and to Tony Swallow at CSIRO for electronics support. We are grateful to A/Prof Brian Chambers of the National Stroke Research Institute (NSRI) for introducing us to the topic of sonothrombolysis and to Dr David Howells of NSRI for guidance on the topic.
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