Fluid flow influences ultrasound-assisted endothelial membrane permeabilization and calcium flux

Ultrasound contrast agent consists of a suspension of encapsulated gas-filled microbubbles, typically polydisperse and ranging in size from 1 to 8 μm in diameter and coated with a compliant biocompatible shell (e.g. phospholipid monolayer) [1]. Microbubbles, including commercially available Definity™, are clinically available and employed in cardiovascular imaging (left ventricular opacification) and recently approved for pediatric imaging [2]. As they remain intravascular due to their size, microbubbles act as red blood cell tracers and significantly enhance the ultrasound signal from within the vascular network [3]. Indeed, as microbubbles vibrate within a focused ultrasound beam, there have been many investigations into their dynamics and scattering behaviour, typically a function of intrinsic factors (e.g. bubble size [4,5], shell constituents [6]) and extrinsic factors (e.g. presence of nearby boundaries [7,8]).

Aside from their diagnostic applications, there has been tremendous progress on using these agents to elicit localized bio-effects; ranging from modulating vascular and cellular permeability [9,10], blood flow [11], local thermal absorption [12], clot lysis [13], cellular signaling [14,15] and neuromodulation [16]. Microbubbles can be made to vibrate with spatial (ultrasound beam focusing, attachment of targeting ligands on the bubble surface [17]) and temporal (ultrasound pulsing scheme) precision and in this sense, present a tremendous opportunity for site-specific modulation of bio-activity. The expansion and contraction of the microbubble within an acoustic field gives rise to local fluid streaming around the bubble [18,19], resulting in shear and circumferential stresses on the vascular wall [20,21]. Of particular interest is the localized enhanced cellular and vascular permeabilization for the purposes of targeted drug and/or gene delivery. Ultrasound-mediated drug/gene delivery as a platform has seen recent success in many applications. Under MRI guidance, focused ultrasound microbubble-mediated blood brain barrier opening [22,23] has been shown to promote the delivery of many formats of molecular therapeutics (e.g. chemotherapeutics [24], adeno-associated virus capsids [25], stem cells [26], NK cells [27], antibodies [28], and cytokines [29]) to targeted regions of the brain that would otherwise be incapable of transiting the cerebrovasculature. The pre-clinical success in this area has led to numerous clinical trials worldwide for enhanced BBB permeability in a variety of pathological contexts (e.g. glioblastoma, Alzheimer's disease). Additionally, while still mostly in the pre-clinical stages, microbubble-mediated therapy has also been shown to enhance drug/gene deposition in non-brain models of cancer (e.g. breast [30], pancreatic [31], carcinomas [32]). Aside from these neurological [33] and oncology-based [34] applications, another major area of investigation is for cardiovascular disease therapy [35], where microbubble-mediated gene therapy has shown promise in a variety of disease states, including cardiomyopathy [36], myocardial infarction [37], ischemia [38], and coronary microembolization [13]. Most recently, there has been a surge of interest in using this technique to modulate the local immune environment for the purposes of immunotherapy [39,40].

Central to the effectiveness of all of these approaches is the oscillation and translational dynamics of microbubbles – particularly those behaviours that lead to the threshold level of stress required to elicit sono-permeabilization [9]. While many investigations have examined salient ultrasound and microbubble factors and how they relate to therapeutic efficiency [[41], [42], [43], [44], [45], [46]], including transmit frequency, peak-negative pressure, sonication time, and microbubble dose, relatively little is known about how the local fluid flow conditions alter microbubble-mediated bio-effects. For a fixed bubble dose (i.e. inter-bubble spacing), ultrasound focal volume, and treatment time, the number of microbubbles that interact with a given set of endothelial cells increases with faster laminar flow. Further, the relative position between a given microbubble and a neighboring cell, and the contact-time of their interaction, is a known factor in the propensity to elicit transient membrane permeability [47]. Given all else equal, the spatial-temporal flow dynamics will greatly affect the proximity of a microbubble to its nearest cell.

Indeed, as the applications of microbubble-mediated drug/gene delivery expand, so too do the local fluid flow conditions under which the microbubbles are situated. In cancer therapy applications, tumor blood vessels are known to be highly irregular in their architecture as compared to those in healthy tissues: geometrically, tumor vessels are heterogeneous in their spatial distribution, dilated and tortuous; and functionally, the abnormal tumor vasculature is characterized by a large number of fenestrae and irregular basement membranes. This elevated geometric and viscous vascular resistance leads to comprised blood flow velocities [48], which can change as a function of tumor type and state of progression [49] and are less dependent on tumor vessel diameter than within healthy tissue. With respect to cardiovascular applications, the severity and extent of ischemia (one of the more investigated cardiovascular applications of microbubble-mediated therapy [35]) will dictate the resulting downstream flow velocity (e.g. [50]), with regions within the centre of the infarcted area exhibiting lower flow than in the peripheral zones [51].

Here, we examine the effects of vascular flow on microbubble-mediated endothelial cell bioeffects using a custom designed acoustically-coupled microscopy system. First, we examine how microbubble velocity effects the propensity of ultrasound-assisted enhanced permeability. Second, we investigate how modification of the ultrasound pulse sequence allows for more effective endothelial cell treatment. Next, we explore how flow rate alters intracellular Ca2+ delivery – a ubiquitous secondary messenger - and its transmission to neighboring, untreated cells. Indeed, Ca2+ plays a pivotal role in living cells, with perhaps its most direct role here being its requirement for plasma membrane repair [52]. However, both intracellular Ca2+ transients and intercellular Ca2+ communication are also well known regulators of many cellular processes, including gene expression and transcriptional regulation [53], endothelial tight junctional contact regulation [54], cell migration and shape [55], and cytokine release [56]. Further, it provides important information on the spatial influence and temporal rate of ultrasound-microbubble treatment on cellular processes. Finally, we discuss the implications of our findings towards modulation of microbubble-assisted drug/gene therapy applications.

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