Ultrasound-Mediated Drug Delivery With a Clinical Ultrasound System: In Vitro Evaluation.

Chemotherapy efficacy is often reduced by insufficient drug uptake in tumor cells. The combination of ultrasound and microbubbles (USMB) has been shown to improve drug delivery and to enhance the efficacy of several drugs in vitro and in vivo, through effects collectively known as sonopermeation. However, clinical translation of USMB therapy is hampered by the large variety of (non-clinical) US set-ups and US parameters that are used in these studies, which are not easily translated to clinical practice. In order to facilitate clinical translation, the aim of this study was to prove that USMB therapy using a clinical ultrasound system (Philips iU22) in combination with clinically approved microbubbles (SonoVue) leads to efficient in vitro sonopermeation. To this end, we measured the efficacy of USMB therapy for different US probes (S5-1, C5-1 and C9-4) and US parameters in FaDu cells. The US probe with the lowest central frequency (i.e. 1.6 MHz for S5-1) showed the highest USMB-induced intracellular uptake of the fluorescent dye SYTOX™ Green (SG). These SG uptake levels were comparable to or even higher than those obtained with a custom-built US system with optimized US parameters. Moreover, USMB therapy with both the clinical and the custom-built US system increased the cytotoxicity of the hydrophilic drug bleomycin. Our results demonstrate that a clinical US system can be used to perform USMB therapy as efficiently as a single-element transducer set-up with optimized US parameters. Therefore, future trials could be based on these clinical US systems, including validated US parameters, in order to accelerate successful translation of USMB therapy.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Microbubble Agents: New Directions.

Microbubble ultrasound contrast agents have now been in use for several decades and their safety and efficacy in a wide range of diagnostic applications have been well established. Recent progress in imaging technology is facilitating exciting developments in techniques such as molecular, 3-D and super resolution imaging and new agents are now being developed to meet their specific requirements. In parallel, there have been significant advances in the therapeutic applications of microbubbles, with recent clinical trials demonstrating drug delivery across the blood-brain barrier and into solid tumours. New agents are similarly being tailored toward these applications, including nanoscale microbubble precursors offering superior circulation times and tissue penetration. The development of novel agents does, however, present several challenges, particularly regarding the regulatory framework. This article reviews the developments in agents for diagnostic, therapeutic and "theranostic" applications; novel manufacturing techniques; and the opportunities and challenges for their commercial and clinical translation.

Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization.

Mistletoe lectin-1 (ML1) is a nature-derived macromolecular cytotoxin that potently induces apoptosis in target cells. Non-specific cytotoxicity to normal cells is one of the major risks in its clinical application, and we therefore propose to encapsulate ML1 in a nanocarrier that can specifically release its cargo intratumorally, thus improving the efficacy to toxicity ratio of the cytotoxin. We investigated the encapsulation of ML1 in ultrasound-sensitive liposomes (USL) and studied its release by high-intensity focused ultrasound (HAccessedIFU). USL were prepared by entrapment of perfluorocarbon nanodroplets in pegylated liposomes. The liposomes were prepared with different DPPC/cholesterol/DSPE-PEG2000 lipid molar ratios (60/20/20 for USL20; 60/30/10 for USL10; 65/30/5 for USL5) before combination with perfluorocarbon (PFC) nanoemulsions (composed of DPPC and perfluoropentane). When triggered with HIFU (peak negative pressure, 2-24 MPa; frequency, 1.3 MHz), PFC nanodroplets can undergo phase transition from liquid to gas thus rupturing the lipid bilayer of usl. Small unilamellar liposomes were obtained with appropriate polydispersity and stability. ML1 and the model protein horseradish peroxidase (HRP) were co-encapsulated with the PFC nanodroplets in USL, with 3% and 7% encapsulation efficiency for USL20 and USL10/USL5, respectively. Acoustic characterization experiments indicated that release is induced by cavitation. HIFU-triggered release of HRP from USL was investigated for optimization of liposomal composition and resulted in 80% triggered release for USL with USL10 (60/30/10) lipid composition. ML1 release from the final USL10 composition was also 80%. Given its high stability, suitable release, and ultrasound sensitivity, USL10 encapsulating ML1 was further used to study released ML1 bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging demonstrated its functional activity regarding the interaction with the target cells. We furthermore demonstrated the cytotoxicity of the released ML1 (I.E., After USL were treated with HIFU). The potent cytotoxicity (IC50 400 ng/ml; free ML1 IC50 345 ng/ml) was compared to non-triggered USL loaded with ML1. Our study shows that USL in combination with HIFU hold promise as trigger-sensitive nanomedicines for local delivery of macromolecular cytotoxins.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.

The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation.

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.

Partial renal coverage in endovascular aneurysm repair causes unfavorable renal flow patterns in an infrarenal aneurysm model.

OBJECTIVE: To achieve an optimal sealing zone during endovascular aneurysm repair, the intended positioning of the proximal end of the endograft fabric should be as close as possible to the most caudal edge of the renal arteries. Some endografts exhibit a small offset between the radiopaque markers and the proximal fabric edge. Unintended partial renal artery coverage may thus occur. This study investigated the consequences of partial coverage on renal flow patterns and wall shear stress (WSS). METHODS: In vitro models of an abdominal aortic aneurysm were used to visualize pulsatile flow using two-dimensional particle image velocimetry under physiologic resting conditions. One model served as control and two models were stented with an Endurant endograft (Medtronic Inc, Minneapolis, Minn), one without and one with partial renal artery coverage with 1.3 mm of stent fabric extending beyond the marker (16% area coverage). The magnitude and oscillation of WSS, relative residence time, and backflow in the renal artery were analyzed. RESULTS: In both stented models, a region along the caudal renal artery wall presented with low and oscillating WSS, not present in the control model. A region with very low WSS (<0.1 Pa) was present in the model with partial coverage over a length of 7 mm compared with a length of 2 mm in the model without renal coverage. Average renal backflow area percentage in the renal artery incrementally increased from control (0.9%) to the stented model without (6.4%) and with renal coverage (18.8%). CONCLUSIONS: In this flow model, partial renal coverage after endovascular aneurysm repair causes low and marked oscillations in WSS, potentially promoting atherosclerosis and subsequent renal artery stenosis. Awareness of the device-dependent offset between the fabric edge and the radiopaque markers is therefore important in endovascular practice.

Laser-driven resonance of dye-doped oil-coated microbubbles: Experimental study.

Photoacoustic (PA) imaging offers several attractive features as a biomedical imaging modality, including excellent spatial resolution and functional information such as tissue oxygenation. A key limitation, however, is the contrast to noise ratio that can be obtained from tissue depths greater than 1-2 mm. Microbubbles coated with an optically absorbing shell have been proposed as a possible contrast agent for PA imaging, offering greater signal amplification and improved biocompatibility compared to metallic nanoparticles. A theoretical description of the dynamics of a coated microbubble subject to laser irradiation has been developed previously. The aim of this study was to test the predictions of the model. Two different types of oil-coated microbubbles were fabricated and then exposed to both pulsed and continuous wave (CW) laser irradiation. Their response was characterized using ultra high-speed imaging. Although there was considerable variability across the population, good agreement was found between the experimental results and theoretical predictions in terms of the frequency and amplitude of microbubble oscillation following pulsed excitation. Under CW irradiation, highly nonlinear behavior was observed which may be of considerable interest for developing different PA imaging techniques with greatly improved contrast enhancement.

Laser-Activated Polymeric Microcapsules for Ultrasound Imaging and Therapy: In Vitro Feasibility.

Polymeric microcapsules with a light-absorbing dye incorporated in their shell can generate vapor microbubbles that can be spatiotemporally controlled by pulsed laser irradiation. These contrast agents of 6-8 µm in diameter can circulate through the vasculature, offering possibilities for ultrasound (molecular) imaging and targeted therapies. Here, we study the impact of such vapor bubbles on human endothelial cells in terms of cell poration and cell viability to establish the imaging and therapeutic windows. Two capsule formulations were used: the first one consisted of a high boiling point oil (hexadecane), whereas the second was loaded with a low boiling point oil (perfluoropentane). Poration probability was already 40% for the smallest bubbles that were formed (<7.5 µm diameter), and reached 100% for the larger bubbles. The hexadecane-loaded capsules also produced bubbles while their shell remained intact. These encapsulated bubbles could therefore be used for noninvasive ultrasound imaging after laser activation without inducing any cell damage. The controlled and localized cell destruction achieved by activation of both capsule formulations may provide an innovative approach for specifically inducing cell death in vivo, e.g., for cancer therapy.

Laser-driven resonance of dye-doped oil-coated microbubbles: A theoretical and numerical study.

Microbubbles are used to enhance the contrast in ultrasound imaging. When coated with an optically absorbing material, these bubbles can also provide contrast in photoacoustic imaging. This multimodal aspect is of pronounced interest to the field of medical imaging. The aim of this paper is to provide a theoretical framework to describe the physical phenomena underlying the photoacoustic response. This article presents a model for a spherical gas microbubble suspended in an aqueous environment and coated with an oil layer containing an optically absorbing dye. The model includes heat transfer between the gas core and the surrounding liquids. This framework is suitable for the investigation of both continuous wave and pulsed laser excitation. This work utilizes a combination of finite difference simulations and numerical integration to determine the dependancy on the physical properties, including composition and thickness of the oil layer on the microbubble response. A normalization scheme for a linearized version of the model was derived to facilitate comparison with experimental measurements. The results show that viscosity and thickness of the oil layer determine whether or not microbubble resonance can be excited. This work also examines the use of non-sinusoidal excitation to promote harmonic imaging techniques to further improve the imaging sensitivity.

Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles.

In the last years, research on ultrasound mediated drug delivery using microbubbles is vastly expanding. While some groups simply mix drugs and microbubbles (co-administration), other researchers have a major interest in the potential of drug-loaded microbubbles. However, today, little is known on the pros and cons of these two strategies. In this study we evaluated the delivery of nanoparticles (polystyrene nanospheres and mRNA-lipoplexes) to cells in vitro, in case the nanoparticles were mixed with unloaded microbubbles versus loaded onto the microbubbles. Flow cytometry experiments demonstrated that unloaded microbubbles did not enhance the cellular delivery of the nanospheres and mRNA-lipoplexes. However, upon loading the nanoparticles onto the microbubbles, their delivery to cells substantially improved. Real-time swept field confocal microscopy imaging of the microbubbles and cells during ultrasound radiation revealed that nanoparticle-loaded microbubbles directly deposited the nanoparticles in patches onto the cell membrane, a process that we termed 'sonoprinting'. This phenomenon resulted in the delivery of large amounts of nanoparticles to the cells and is suggested to be different from the creation of cell membrane pores and enhanced endocytosis, which have been reported before as mechanisms behind the improved delivery of drugs to cells by ultrasound.