Sonosensitive Cavitation Nuclei-A Customisable Platform Technology for Enhanced Therapeutic Delivery.

Ultrasound-mediated cavitation shows great promise for improving targeted drug delivery across a range of clinical applications. Cavitation nuclei-sound-sensitive constructs that enhance cavitation activity at lower pressures-have become a powerful adjuvant to ultrasound-based treatments, and more recently emerged as a drug delivery vehicle in their own right. The unique combination of physical, biological, and chemical effects that occur around these structures, as well as their varied compositions and morphologies, make cavitation nuclei an attractive platform for creating delivery systems tuned to particular therapeutics. In this review, we describe the structure and function of cavitation nuclei, approaches to their functionalization and customization, various clinical applications, progress toward real-world translation, and future directions for the field.

An Acoustic Device for Ultra High-Speed Quantification of Cell Strain During Cell-Microbubble Interaction.

Microbubbles utilize high-frequency oscillations under ultrasound stimulation to induce a range of therapeutic effects in cells, often through mechanical stimulation and permeabilization of cells. One of the largest challenges remaining in the field is the characterization of interactions between cells and microbubbles at therapeutically relevant frequencies. Technical limitations, such as employing sufficient frame rates and obtaining sufficient image resolution, restrict the quantification of the cell's mechanical response to oscillating microbubbles. Here, a novel methodology was developed to address many of these limitations and improve the image resolution of cell-microbubble interactions at high frame rates. A compact acoustic device was designed to house cells and microbubbles as well as a therapeutically relevant acoustic field while being compatible with a Shimadzu HPV-X camera. Cell viability tests confirmed the successful culture and proliferation of cells, and the attachment of DSPC- and cationic DSEPC-microbubbles to osteosarcoma cells was quantified. Microbubble oscillation was observed within the device at a frame rate of 5 million FPS, confirming suitable acoustic field generation and ultra high-speed image capture. High spatial resolution in these images revealed observable deformation in cells following microbubble oscillation and supported the first use of digital image correlation for strain quantification in a single cell. The novel acoustic device provided a simple, effective method for improving the spatial resolution of cell-microbubble interaction images, presenting the opportunity to develop an understanding of the mechanisms driving the therapeutic effects of oscillating microbubbles upon ultrasound exposure.

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms.

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Investigation of the Phase of Nonlinear Echoes From Microbubbles During Amplitude Modulation.

Contrast-enhanced ultrasound (CEUS) imaging relies on distinguishing between microbubble and tissue echoes. Amplitude modulation (AM), a nonlinear pulsing scheme, has been developed to take advantage of the amplitude-dependent nonlinearity of microbubble echoes. However, with AM, tissue nonlinear propagation can also degrade the image contrast. Segmentation of CEUS images based on amplitude-dependent phase difference in the echoes, defined in this article as [Formula: see text], has been proposed as an additional method of enhancing contrast-to-tissue ratio as tissue is not expected to create the same degree of [Formula: see text]; however, this has not been robustly investigated. In this work, we evaluate the source of [Formula: see text] through simulations of unshelled versus shelled microbubble oscillation and simulations of nonlinear propagation in tissue. We then validate the simulated [Formula: see text] results with experimental [Formula: see text] measurements during in vitro scattering and imaging in a flow phantom. We show that shelled and unshelled microbubbles resulted in a [Formula: see text] with similar overall magnitude with some differences in trends, and that tissue echoes have a small yet detectable degree of [Formula: see text] due to nonlinear propagation. The results from this work can help inform optimal parameter selection for phase segmentation and implementation on a clinical scanner.

The Role of Ultrasound in Modulating Interstitial Fluid Pressure in Solid Tumors for Improved Drug Delivery.

The unique microenvironment of solid tumors, including desmoplasia within the extracellular matrix, enhanced vascular permeability, and poor lymphatic drainage, leads to an elevated interstitial fluid pressure which is a major barrier to drug delivery. Reducing tumor interstitial fluid pressure is one proposed method of increasing drug delivery to the tumor. The goal of this topical review is to describe recent work using focused ultrasound with or without microbubbles to modulate tumor interstitial fluid pressure, through either thermal or mechanical effects on the extracellular matrix and the vasculature. Furthermore, we provide a review on techniques in which ultrasound imaging may be used to diagnose elevated interstitial fluid pressure within solid tumors. Ultrasound-based techniques show high promise in diagnosing and treating elevated interstitial pressure to enhance drug delivery.

Characterizing Intracranial Hemodynamics in Sickle Cell Anemia: Impact of Patient-Specific Viscosity.

PURPOSE: Pediatric and adult patients with sickle cell anemia (SCA) are at increased risk of stroke and cerebrovascular accident. In the general adult population, there is a relationship between arterial hemodynamics and pathology; however, this relationship in SCA patients remains to be elucidated. The aim of this work was to characterize circle of Willis hemodynamics in patients with SCA and quantify the impact of viscosity choice on pathophysiologically-relevant hemodynamics measures. METHODS: Based on measured vascular geometries, time-varying flow rates, and blood parameters, detailed patient-specific simulations of the circle of Willis were conducted for SCA patients (n = 6). Simulations quantified the impact of patient-specific and standard blood viscosities on wall shear stress (WSS). RESULTS: These results demonstrated that use of a standard blood viscosity introduces large errors into the estimation of pathophysiologically-relevant hemodynamic parameters. Standard viscosity models overpredicted peak WSS by 55% and 49% for steady and pulsatile flow, respectively. Moreover, these results demonstrated non-uniform, spatial patterns of positive and negative WSS errors related to viscosity, and standard viscosity simulations overpredicted the time-averaged WSS by 32% (standard deviation = 7.1%). Finally, differences in shear rate demonstrated that the viscosity choice alters the simulated near-wall flow field, impacting hemodynamics measures. CONCLUSIONS: This work presents simulations of circle of Willis arterial flow in SCA patients and demonstrates the importance and feasibility of using a patient-specific viscosity in these simulations. Accurately characterizing cerebrovascular hemodynamics in SCA populations has potential for elucidating the pathophysiology of large-vessel occlusion, aneurysms, and tissue damage in these patients.

Safety of Image-Guided Treatment of the Liver with Ultrasound and Microbubbles in an in Vivo Porcine Model.

Ultrasound and microbubbles are useful for both diagnostic imaging and targeted drug delivery, making them ideal conduits for theranostic interventions. Recent reports have indicated the preclinical success of microbubble cavitation for enhancement of chemotherapy in abdominal tumors; however, there have been limited studies and variable efficacy in clinical implementation of this technique. This is likely because in contrast to the high pressures and long cycle lengths seen in successful preclinical work, current clinical implementation of microbubble cavitation for drug delivery generally involves low acoustic pressures and short cycle lengths to fit within clinical guidelines. To translate the preclinical parameter space to clinical adoption, a relevant safety study in a healthy large animal is required. Therefore, the purpose of this work was to evaluate the safety of ultrasound cavitation treatment (USCTx) in a healthy porcine model using a modified Philips EPIQ with S5-1 as the focused source. We performed USCTx on eight healthy pigs and monitored health over the course of 1 wk. We then performed an acute study of USCTx to evaluate immediate tissue damage. Contrast-enhanced ultrasound exams were performed before and after each treatment to investigate perfusion changes within the treated areas, and blood and urine were evaluated for liver damage biomarkers. We illustrate, through quantitative analysis of contrast-enhanced ultrasound data, blood and urine analyses and histology, that this technique and the parameter space considered are safe within the time frame evaluated. With its safety confirmed using a clinical-grade ultrasound scanner and contrast agent, USCTx could be easily translated into clinical trials for improvement of chemotherapy delivery. This represents the first safety study assessing the bio-effects of microbubble cavitation from relevant ultrasound parameters in a large animal model.

Cavitation Therapy Monitoring of Commercial Microbubbles With a Clinical Scanner.

The ability to monitor cavitation activity during ultrasound and microbubble-mediated procedures is of high clinical value. However, there has been little reported literature comparing the cavitation characteristics of different clinical microbubbles, nor have current clinical scanners been used to perform passive cavitation detection in real time. The goal of this work was to investigate and characterize standard microbubble formulations (Optison, Sonovue, Sonazoid, and a custom microbubble made with similar components as Definity) with a custom passive cavitation detector (two confocal single-element focused transducers) and with a Philips EPIQ scanner with a C5-1 curvilinear probe passively listening. We evaluated three different methods for investigating cavitation thresholds, two from previously reported work and one developed in this work. For all three techniques, it was observed that the inertial cavitation thresholds were between 0.1 and 0.3 MPa for all agents when detected with both systems. Notably, we found that most microbubble formulations in bulk solution behaved generally similarly, with some differences. We show that these characteristics and thresholds are maintained when using a diagnostic ultrasound system for detecting cavitation activity. We believe that a systematic evaluation of the frequency response of the cavitation activity of different microbubbles in order to inform real-time therapy monitoring using a clinical ultrasound device could make an immediate clinical impact.

Image-Guided Treatment of Primary Liver Cancer in Mice Leads to Vascular Disruption and Increased Drug Penetration.

Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the fourth leading cause of cancer-related deaths worldwide with a <30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many treatment options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound-induced cavitation can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided, cavitation-enabled drug delivery with a clinical ultrasound scanner. To this end, ultrasound conditions (unique from those used in imaging) were designed and implemented on a Philips EPIQ and S5-1 phased array probe to produced focused ultrasound for cavitation treatment. Sonovue® microbubbles which are clinically approved as an ultrasound contrast agent were used for both imaging and cavitation treatment. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that image-guided and targeted microbubble cavitation resulted in selective disruption of the tumor blood flow and enhanced doxorubicin uptake and penetration. Histology results indicate that no gross morphological damage occurred as a result of this process. The combination of these effects may be exploited to treat HCC and other challenging malignancies and could be implemented with currently available ultrasound scanners and reagents.

Engineered 3D Microvascular Networks for the Study of Ultrasound-Microbubble-Mediated Drug Delivery.

Localized and targeted drug delivery can be achieved by the combined action of ultrasound and microbubbles on the tumor microenvironment, likely through sonoporation and other therapeutic mechanisms that are not well understood. Here, we present a perfusable in vitro model with a realistic 3D geometry to study the interactions between microbubbles and the vascular endothelium in the presence of ultrasound. Specifically, a three-dimensional, endothelial-cell-seeded in vitro microvascular model was perfused with cell culture medium and microbubbles while being sonicated by a single-element 1 MHz focused transducer. This setup mimics the in vivo scenario in which ultrasound induces a therapeutic effect in the tumor vasculature in the presence of flow. Fluorescence and bright-field microscopy were employed to assess the microbubble-vessel interactions and the extent of drug delivery and cell death both in real time during treatment as well as after treatment. Propidium iodide was used as the model drug while calcein AM was used to evaluate cell viability. There were two acoustic parameter sets chosen for this work: (1) acoustic pressure: 1.4 MPa, pulse length: 500 cycles, duty cycle: 5% and (2) acoustic pressure: 0.4 MPa, pulse length: 1000 cycles, duty cycle: 20%. Enhanced drug delivery and cell death were observed in both cases while the higher pressure setting had a more pronounced effect. By introducing physiological flow to the in vitro microvascular model and examining the PECAM-1 expression of the endothelial cells within it, we demonstrated that our model is a good mimic of the in vivo vasculature and is therefore a viable platform to provide mechanistic insights into ultrasound-mediated drug delivery.

Combined In Silico and In Vitro Approach Predicts Low Wall Shear Stress Regions in a Hemofilter that Correlate with Thrombus Formation In Vivo.

A major challenge in developing blood-contacting medical devices is mitigating thrombogenicity of an intravascular device. Thrombi may interfere with device function or embolize from the device to occlude distant vascular beds with catastrophic consequences. Chemical interactions between plasma proteins and bioengineered surface occur at the nanometer scale; however, continuum models of blood predict local shear stresses that lead to platelet activation or aggregation and thrombosis. Here, an iterative approach to blood flow path design incorporating in silico, in vitro, and in vivo experiments predicted the occurrence and location of thrombi in an implantable hemofilter. Low wall shear stress (WSS) regions identified by computational fluid dynamics (CFD) predicted clot formation in vivo. Revised designs based on CFD demonstrated superior performance, illustrating the importance of a multipronged approach for a successful design process.