A drug-selectable acoustic reporter gene system for human cell ultrasound imaging.

A promising new field of genetically encoded ultrasound contrast agents in the form of gas vesicles has recently emerged, which could extend the specificity of medical ultrasound imaging. However, given the delicate genetic nature of how these genes are integrated and expressed, current methods of producing gas vesicle-expressing mammalian cell lines requires significant cell processing time to establish a clonal/polyclonal line that robustly expresses the gas vesicles sufficiently enough for ultrasound contrast. Here, we describe an inducible and drug-selectable acoustic reporter gene system that can enable gas vesicle expression in mammalian cell lines, which we demonstrate using HEK293T cells. Our drug-selectable construct design increases the stability and proportion of cells that successfully integrate all plasmids into their genome, thus reducing the amount of cell processing time required. Additionally, we demonstrate that our drug-selectable strategy forgoes the need for single-cell cloning and fluorescence-activated cell sorting, and that a drug-selected mixed population is sufficient to generate robust ultrasound contrast. Successful gas vesicle expression was optically and ultrasonically verified, with cells expressing gas vesicles exhibiting an 80% greater signal-to-noise ratio compared to negative controls and a 500% greater signal-to-noise ratio compared to wild-type HEK293T cells. This technology presents a new reporter gene paradigm by which ultrasound can be harnessed to visualize specific cell types for applications including cellular reporting and cell therapies.

A Model of High-Speed Endovascular Sonothrombolysis with Vortex Ultrasound-Induced Shear Stress to Treat Cerebral Venous Sinus Thrombosis.

This research aims to demonstrate a novel vortex ultrasound enabled endovascular thrombolysis method designed for treating cerebral venous sinus thrombosis (CVST). This is a topic of substantial importance since current treatment modalities for CVST still fail in as many as 20% to 40% of the cases, and the incidence of CVST has increased since the outbreak of the coronavirus disease 2019 pandemic. Compared with conventional anticoagulant or thrombolytic drugs, sonothrombolysis has the potential to remarkably shorten the required treatment time owing to the direct clot targeting with acoustic waves. However, previously reported strategies for sonothrombolysis have not demonstrated clinically meaningful outcomes (e.g., recanalization within 30 min) in treating large, completely occluded veins or arteries. Here, we demonstrated a new vortex ultrasound technique for endovascular sonothrombolysis utilizing wave-matter interaction-induced shear stress to enhance the lytic rate substantially. Our in vitro experiment showed that the lytic rate was increased by at least 64.3% compared with the nonvortex endovascular ultrasound treatment. A 3.1-g, 7.5-cm-long, completely occluded in vitro 3-dimensional model of acute CVST was fully recanalized within 8 min with a record-high lytic rate of 237.5 mg/min for acute bovine clot in vitro. Furthermore, we confirmed that the vortex ultrasound causes no vessel wall damage over ex vivo canine veins. This vortex ultrasound thrombolysis technique potentially presents a new life-saving tool for severe CVST cases that cannot be efficaciously treated using existing therapies.

Perfluorocarbon nanodroplet size, acoustic vaporization, and inertial cavitation affected by lipid shell composition in vitro.

Perfluorocarbon nanodroplets (PFCnDs) are ultrasound contrast agents that phase-transition from liquid nanodroplets to gas microbubbles when activated by laser irradiation or insonated with an ultrasound pulse. The dynamics of PFCnDs can vary drastically depending on the nanodroplet composition, including the lipid shell properties. In this paper, we investigate the effect of varying the ratio of PEGylated to non-PEGylated phospholipids in the outer shell of PFCnDs on the acoustic nanodroplet vaporization (liquid to gas phase transition) and inertial cavitation (rapid collapse of the vaporized nanodroplets) dynamics in vitro when insonated with focused ultrasound. Nanodroplets with a high concentration of PEGylated lipids had larger diameters and exhibited greater variance in size distribution compared to nanodroplets with lower proportions of PEGylated lipids in the lipid shell. PFCnDs with a lipid shell composed of 50:50 PEGylated to non-PEGylated lipids yielded the highest B-mode image intensity and duration, as well as the greatest pressure difference between acoustic droplet vaporization onset and inertial cavitation onset. We demonstrate that slight changes in lipid shell composition of PFCnDs can significantly impact droplet phase transitioning and inertial cavitation dynamics. These findings can help guide researchers to fabricate PFCnDs with optimized compositions for their specific applications.

Extreme material parameters accessible by active acoustic metamaterials with Willis coupling.

Active acoustic metamaterials incorporate electric circuit elements that input energy into an otherwise passive medium to aptly modulate the effective material properties. Here, we propose an active acoustic metamaterial with Willis coupling to drastically extend the tunability of the effective density and bulk modulus with the accessible parameter range enlarged by at least two orders of magnitude compared to that of a non-Willis metamaterial. Traditional active metamaterial designs are based on local resonances without considering the Willis coupling that limit their accessible effective material parameter range. Our design adopts a unit cell structure with two sensor-transducer pairs coupling the acoustic response on both sides of the metamaterial by detecting incident waves and driving active signals asymmetrically superimposed onto the passive response of the material. The Willis coupling results from feedback control circuits with unequal gains. These asymmetric feedback control circuits use Willis coupling to expand the accessible range of the effective density and bulk modulus of the metamaterial. The extreme effective material parameters realizable by the metamaterials will remarkably broaden their applications in biomedical imaging, noise control, and transformation acoustics-based cloaking.