Cardiac gene delivery using ultrasound: State of the field.

Over the past two decades, there has been tremendous and exciting progress toward extending the use of medical ultrasound beyond a traditional imaging tool. Ultrasound contrast agents, typically used for improved visualization of blood flow, have been explored as novel non-viral gene delivery vectors for cardiovascular therapy. Given this adaptation to ultrasound contrast-enhancing agents, this presents as an image-guided and site-specific gene delivery technique with potential for multi-gene and repeatable delivery protocols-overcoming some of the limitations of alternative gene therapy approaches. In this review, we provide an overview of the studies to date that employ this technique toward cardiac gene therapy using cardiovascular disease animal models and summarize their key findings.

Shear stress preconditioning and microbubble flow pattern modulate ultrasound-assisted plasma membrane permeabilization.

The recent and exciting success of anti-inflammatory therapies for ischemic heart disease (e.g. atherosclerosis) is hindered by the lack of site-specific and targeted therapeutic deposition. Microbubble-mediated focused ultrasound, which uses circulating, lipid-encapsulated intravascular microbubbles to locally enhance endothelial permeability, offers an exciting approach. Atherosclerotic plaques preferentially develop in regions with disturbed blood flow, and microbubble-endothelial cell membrane interactions under such flow conditions are not well understood. Here, using an acoustically-coupled microscopy system, endothelial cells were sonicated (1 MHz, 20 cycle bursts, 1 ms PRI, 4 s duration, 300 kPa peak-negative pressure) under perfusion with Definity™ bubbles to examine microbubble-mediated endothelial permeabilization under a range of physiological conditions. Endothelial preconditioning under prolonged shear influenced physiology and the secretome, inducing increased expression of pro-angiogenesis analytes, decreasing levels of pro-inflammatory ones, and increasing the susceptibility of ultrasound therapy. Ultrasound treatment efficiency was positively correlated with concentrations of pro-angiogenic cytokines (e.g. VEGF-A, EGF, FGF-2), and negatively correlated with pro-inflammatory chemokines (e.g. MCP-1, GCP-2, SDF-1). Furthermore, ultrasound therapy under non-reversing pulsatile flow (∼4-8 dyne/cm2, 0.5-1 Hz) increased permeabilization up to 2.4-fold compared to shear-matched laminar flow, yet treatment under reversing oscillatory flow resulted in more heterogeneous modulation. This study provides insight into the role of vascular physiology, including endothelial biology, into the design of a localized ultrasound drug delivery system for ischemic heart disease.

Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells (SCC-VII-Luc) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase (Luc siRNA) were suspended. Ultrasound (US) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice (n=23) received either EGFR siRNA-loaded LPX + UTMC or negative control (NC) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p<0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p<0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro. EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo, suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.

ULTRA-SR Challenge: Assessment of Ultrasound Localization and TRacking Algorithms for Super-Resolution Imaging.

With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.

Subharmonic resonance of phospholipid coated ultrasound contrast agent microbubbles.

Phospholipid encapsulated ultrasound contrast agents have proven to be a powerful addition in diagnostic imaging and show emerging applications in targeted therapy due to their resonant and nonlinear scattering. Microbubble response is affected by their intrinsic (e.g. bubble size, encapsulation physics) and extrinsic (e.g. boundaries) factors. One of the major intrinsic factors at play affecting microbubble vibration dynamics is the initial phospholipid packing of the lipid encapsulation. Here, we examine how the initial phospholipid packing affects the subharmonic response of either individual or a system of two closely-placed microbubbles. We employ a finite element model to investigate the change in subharmonic resonance under 'small' and 'large' radial excursions. For microbubbles ranging between 1.5 and 2.5 µm in diameter and in its elastic state (σ0 = 0.01 N/m), we demonstrate up to a 10 % shift towards lower frequencies in the peak subharmonic response as the radial excursion increases. However, for a bubble initially in its buckled state (σ0 = 0 N/m), we observe a maximum shift of 8 % towards higher frequencies as the radial excursion increases over the same range of bubble sizes - the opposite trend. We studied the same scenario for a system of two individual microbubbles for which we saw similar results. For microbubbles that are initially in their elastic state, in both cases of a) two identically sized bubbles and b) a bubble in proximity to a smaller bubble, we observed a 6 % and 9 % shift towards lower frequencies respectively; while in the case of a neighboring larger bubble no change in subharmonic resonance frequency was observed. Microbubbles that are initially in a buckled state exert no change, 5 % and 19 % shift towards higher frequencies, in two-bubble systems consisting of a) same-size, b) smaller, and c) larger neighboring bubble respectively. Furthermore, we examined the effect of two adjacent bubbles with non-equal initial phospholipid states. The results presented here have important implications in ultrasound contrast agent applications.

Focused ultrasound-assisted delivery of immunomodulating agents in brain cancer.

Focused ultrasound (FUS) combined with intravascularly circulating microbubbles can transiently increase the permeability of the blood-brain barrier (BBB) to enable targeted therapeutic delivery to the brain, the clinical testing of which is currently underway in both adult and pediatric patients. Aside from traditional cancer drugs, this technique is being extended to promote the delivery of immunomodulating therapeutics to the brain, including antibodies, immune cells, and cytokines. In this manner, FUS approaches are being explored as a tool to improve and amplify the effectiveness of immunotherapy for both primary and metastatic brain cancer, a particularly challenging solid tumor to treat. Here, we present an overview of the latest groundbreaking research in FUS-assisted delivery of immunomodulating agents to the brain in pre-clinical models of brain cancer, and place it within the context of the current immunotherapy approaches. We follow this up with a discussion on new developments and emerging strategies for this rapidly evolving approach.

Cavitation-Enhanced Drug Delivery and Immunotherapy.

Welcome to this special issue on Cavitation-Enhanced Drug Delivery and Immunotherapy-a rapidly evolving area that has been buoyed in recent years by the development of methods harnessing the activity of ultrasound-stimulated bubbles known as cavitation [...].

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

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30 ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1 MHz ultrasound with 20-cycle bursts, 1 ms PRI, and 2 s duration at a peak negative pressure of 305 kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t = 1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.

Stable Cavitation-Mediated Delivery of miR-126 to Endothelial Cells.

In endothelial cells, microRNA-126 (miR-126) promotes angiogenesis, and modulating the intracellular levels of this gene could suggest a method to treat cardiovascular diseases such as ischemia. Novel ultrasound-stimulated microbubbles offer a means to deliver therapeutic payloads to target cells and sites of disease. The purpose of this study was to investigate the feasibility of gene delivery by stimulating miR-126-decorated microbubbles using gentle acoustic conditions (stable cavitation). A cationic DSTAP microbubble was formulated and characterized to carry 6 µg of a miR-126 payload per 109 microbubbles. Human umbilical vein endothelial cells (HUVECs) were treated at 20−40% duty cycle with miR-126-conjugated microbubbles in a custom ultrasound setup coupled with a passive cavitation detection system. Transfection efficiency was assessed by RT-qPCR, Western blotting, and endothelial tube formation assay, while HUVEC viability was monitored by MTT assay. With increasing duty cycle, the trend observed was an increase in intracellular miR-126 levels, up to a 2.3-fold increase, as well as a decrease in SPRED1 (by 33%) and PIK3R2 (by 46%) expression, two salient miR-126 targets. Under these ultrasound parameters, HUVECs maintained >95% viability after 96 h. The present work describes the delivery of a proangiogenic miR-126 using an ultrasound-responsive cationic microbubble with potential to stimulate therapeutic angiogenesis while minimizing endothelial damage.

The influence of inter-bubble spacing on the resonance response of ultrasound contrast agent microbubbles.

Ultrasound-driven microbubbles, typically between 1 and 8 µm in diameter, are resonant scatterers that are employed as diagnostic contrast agents and emerging as potentiators of targeted therapies. Microbubbles are administered in populations whereby their radial dynamics - key to their effectiveness - are greatly affected by intrinsic (e.g. bubble size) and extrinsic (e.g. boundaries) factors. In this work, we aim to understand how two neighbouring microbubbles influence each other. We developed a finite element model of a system of two individual phospholipid-encapsulated microbubbles vibrating in proximity to each other to study the effect of inter-bubble distance on microbubble radial resonance response. For the case of two equal-sized and identical bubbles, each bubble exhibits a decrease between 7 and 10% in the frequency of maximum response (fMR) and an increase in amplitude of maximum response (AMR) by 9-11% as compared to its isolated response in free-space, depending on the bubble size examined. For a system of two unequal-sized microbubbles, the large bubble shows no significant change, however the smaller microbubble shows an increase in fMR by 7-11% and a significant decrease in AMR by 38-52%. Furthermore, in very close proximity the small bubble shows a secondary off-resonance peak at the corresponding fMR of its larger companion microbubble. Our work suggests that frequency-dependent microbubble response is greatly affected by the presence of another bubble, which has implications in both imaging and therapy applications. Furthermore, our work suggests a mechanism by which nanobubbles show significant off-resonance vibrations in the clinical frequency range, a behaviour that has been observed experimentally but heretofore unexplained.

An Overview of Cell Membrane Perforation and Resealing Mechanisms for Localized Drug Delivery.

Localized and reversible plasma membrane disruption is a promising technique employed for the targeted deposition of exogenous therapeutic compounds for the treatment of disease. Indeed, the plasma membrane represents a significant barrier to successful delivery, and various physical methods using light, sound, and electrical energy have been developed to generate cell membrane perforations to circumvent this issue. To restore homeostasis and preserve viability, localized cellular repair mechanisms are subsequently triggered to initiate a rapid restoration of plasma membrane integrity. Here, we summarize the known emergency membrane repair responses, detailing the salient membrane sealing proteins as well as the underlying cytoskeletal remodeling that follows the physical induction of a localized plasma membrane pore, and we present an overview of potential modulation strategies that may improve targeted drug delivery approaches.

Lateral Position-Dependent Velocity Estimation Error in Plane-Wave Doppler Ultrasound Systems.

Doppler ultrasound has become a standard method used to diagnose and grade vascular diseases and monitor their progression. Conventional focused-beam color Doppler imaging is routinely used in clinical practice, but suffers from inherent trade-offs between spatial, temporal and velocity resolution. Newer, plane-wave Doppler imaging offers rapid simultaneous acquisition of B-mode, color and spectral Doppler information across large fields of view, making it a potentially useful method for quantitative estimation of blood flow velocities in the clinic. However, plane-wave imaging can lead to a substantial error in velocity estimation, which is dependent on the lateral location within the image. This is seen in both clinical and experimental plane-wave systems. In the work described in this article, we quantified this velocity error under different geometric and beamforming conditions using numerical simulation and experimental phantoms. We found that the lateral-dependent velocity errors are caused by asymmetrical geometric spectral broadening, and outline a correction algorithm that can mitigate these errors.

Investigating the Accumulation of Submicron Phase-Change Droplets in Tumors.

Submicron phase-change droplets are an emerging class of ultrasound contrast agent. Compared with microbubbles, their relatively small size and increased stability offer the potential to passively extravasate and accumulate in solid tumors through the enhanced permeability and retention effect. Under exposure to sufficiently powerful ultrasound, these droplets can convert into in situ gas microbubbles and thus be used as an extravascular-specific contrast agent. However, in vivo imaging methods to detect extravasated droplets have yet to be established. Here, we develop an ultrasound imaging pulse sequence within diagnostic safety limits to selectively detect droplet extravasation in tumors. Tumor-bearing mice were injected with submicron perfluorobutane droplets and interrogated with our imaging-vaporization-imaging sequence. By use of a pulse subtraction method, median droplet extravasation signal relative to the total signal within the tumor was estimated to be Etumor=37±5% compared with the kidney Ekidney=-2±8% (p < 0.001). This work contributes toward the advancement of volatile phase-shift droplets as a next-generation ultrasound agent for imaging and therapy.

Transendothelial Perforations and the Sphere of Influence of Single-Site Sonoporation.

Acoustically driven gas bubble cavitation locally concentrates energy and can result in physical phenomena including sonoluminescence and erosion. In biomedicine, ultrasound-driven microbubbles transiently increase plasma membrane permeability (sonoporation) to promote drug/gene delivery. Despite its potential, little is known about cellular response in the aftermath of sonoporation. In the work described here, using a live-cell approach, we assessed the real-time interplay between transendothelial perforations (∼30-60 s) up to 650 µm2, calcium influx, breaching of the local cytoskeleton and sonoporation resealing upon F-actin recruitment to the perforation site (∼5-10 min). Through biophysical modeling, we established the critical role of membrane line tension in perforation resealing velocity (10-30 nm/s). Membrane budding/shedding post-sonoporation was observed on complete perforation closure, yet successful pore repair does not mark the end of sonoporation: protracted cell mobility from 8 µs of ultrasound is observed up to 4 h post-treatment. Taken holistically, we established the biophysical context of endothelial sonoporation repair with application in drug/gene delivery.

A Review of Phospholipid Encapsulated Ultrasound Contrast Agent Microbubble Physics.

Ultrasound contrast agent microbubbles have expanded the utility of biomedical ultrasound from anatomic imaging to the assessment of microvascular blood flow characteristics and ultrasound-assisted therapeutic applications. Central to their effectiveness in these applications is their resonant and non-linear oscillation behaviour. This article reviews the salient physics of an oscillating microbubble in an ultrasound field, with particular emphasis on phospholipid-coated agents. Both the theoretical underpinnings of bubble vibration and the experimental evidence of non-linear encapsulated bubble dynamics and scattering are discussed and placed within the context of current and emerging applications.

Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles.

Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales-with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability-bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.

Biophysical insight into mechanisms of sonoporation.

This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity.

Ultrasound-stimulated microbubble dynamics have been shown to be dependent on intrinsic bubble properties, including size and shell characteristics. The effect of the surrounding environment on microbubble response, however, has been less investigated. In particular, microbubble optimization studies are generally conducted in water/saline, characterized by a 1 cP viscosity, for application in the vasculature (i.e., 4 cP). In this study, ultra-high speed microscopy was employed to investigate fluid viscosity effects on phospholipid encapsulated microbubble oscillations at 1 MHz, using a single, eight-cycle pulse at peak negative pressures of 100 and 250 kPa. Microbubble oscillations were shown to be affected by fluid viscosity in a size- and pressure-dependent manner. In general, the oscillation amplitudes exhibited by microbubbles between 3 and 6 µm in 1 cP fluid were larger than in 4 cP fluid, reaching a maximum of 1.7-fold at 100 kPa for microbubbles 3.8 µm in diameter and 1.35-fold at 250 kPa for microbubbles 4.8 µm in diameter. Simulation results were in broad agreement at 250 kPa, however generally underestimated the effect of fluid viscosity at 100 kPa. This is the first experimental demonstration documenting the effects of surrounding fluid viscosity on microbubble oscillations, resulting in behavior not entirely predicted by current microbubble models.

Fluid Viscosity Affects the Fragmentation and Inertial Cavitation Threshold of Lipid-Encapsulated Microbubbles.

Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (∼4 cP). In this study, ultrahigh-speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25-2 MPa). The propensity for individual microbubble (n = 220) fragmentation was found to significantly decrease in 4-cP fluid compared with 1-cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4-cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g., ultraharmonic) with increasing pressure, compared with those in 1-cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations.

In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging.

Converting nanoparticles or monomeric compounds into larger supramolecular structures by endogenous or external stimuli is increasingly popular because these materials are useful for imaging and treating diseases. However, conversion of microstructures to nanostructures is less common. Here, we show the conversion of microbubbles to nanoparticles using low-frequency ultrasound. The microbubble consists of a bacteriochlorophyll-lipid shell around a perfluoropropane gas. The encapsulated gas provides ultrasound imaging contrast and the porphyrins in the shell confer photoacoustic and fluorescent properties. On exposure to ultrasound, the microbubbles burst and form smaller nanoparticles that possess the same optical properties as the original microbubble. We show that this conversion is possible in tumour-bearing mice and could be validated using photoacoustic imaging. With this conversion, our microbubble can potentially be used to bypass the enhanced permeability and retention effect when delivering drugs to tumours.