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

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

HIGH INTENSITY FOCUSED ULTRASOUND AND OXYGEN LOAD NANOBUBBLES: TWO DIFFERENT APPROCHES FOR CANCER TREATMENT

The study of applications based on the use of ultrasound in medicine and biology for therapeutic purposes is under strong development at international level and joins the notoriously well-established and widespread use of diagnostic applications [1]. In the past few years, High Intensity Focused Ultrasound (HIFU) has developed from a scientific curiosity to an accepted therapeutic modality. HIFU is a non invasive technique for the treatment of various types of cancer, as well as non-malignant pathologies, by inducing localized hyperthermia that causes necrosis of the tissue. Beside HIFU technology, other innovative therapeutic modalities to treat cancer are emerging. Among them, an extremely innovative technique is represented by oxygen loaded nanobubbles (OLNs): gas cavities confined by an appropriately functionalized coating. This is an oxygenating drugs aimed at re-oxygenation of cancerous tissue. Oxygen deficiency, in fact, is the main hallmark of cancerous solid tumors and a major factor limiting the effectiveness of radiotherapy. In this work, these two approaches to treat tumours are under study from a metrological point of view. In particular, a complete characterization of an HIFU fields regarding power, pressure and temperature is provided while oxygen load nanobubbles are synthesized, characterized and applied in in vitro and in vivo experiments

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

An Ultrasonically Actuated Needle Promotes the Transport of Nanoparticles and Fluids

Non-invasive therapeutic ultrasound methods, such as high-intensity focused ultrasound (HIFU), have limited access to tissue targets shadowed by bones or presence of gas. This study demonstrates that an ultrasonically actuated medical needle can be used to translate nanoparticles and fluids under the action of nonlinear phenomena, potentially overcoming some limitations of HIFU. A simulation study was first conducted to study the delivery of a tracer with an ultrasonically actuated needle (33 kHz) inside a porous medium acting as a model for soft tissue. The model was then validated experimentally in different concentrations of agarose gel showing a close match with the experimental results, when diluted soot nanoparticles (diameter < 150 nm) were employed as delivered entity. An additional simulation study demonstrated a threefold increase of the volume covered by the delivered agent in liver under a constant injection rate, when compared to without ultrasound. This method, if developed to its full potential, could serve as a cost effective way to improve safety and efficacy of drug therapies by maximizing the concentration of delivered entities within e.g. a small lesion, while minimizing exposure outside the lesion.Comment: 34 pages, 4 figures, under review in the Journal of the Acoustical Society of Americ

Sono-processes:Emerging systems and their applicability within the (bio-)medical field

Sonochemistry, although established in various fields, is still an emerging field finding new effects of ultrasound on chemical systems and are of particular interest for the biomedical field. This interdisciplinary area of research explores the use of acoustic waves with frequencies ranging from 20 kHz to 1 MHz to induce physical and chemical changes. By subjecting liquids to ultrasonic waves, sonochemistry has demonstrated the ability to accelerate reaction rates, alter chemical reaction pathways, and change physical properties of the system while operating under mild reaction conditions. It has found its way into diverse industries including food processing, pharmaceuticals, material science, and environmental remediation. This review provides an overview of the principles, advancements, and applications of sonochemistry with a particular focus on the domain of (bio-)medicine. Despite the numerous benefits sonochemistry has to offer, most of the research in the (bio-)medical field remains in the laboratory stage. Translation of these systems into clinical practice is complex as parameters used for medical ultrasound are limited and toxic side effects must be minimized in order to meet regulatory approval. However, directing attention towards the applicability of the system in clinical practice from the early stages of research holds significant potential to further amplify the role of sonochemistry in clinical applications.</p

Vibration-Induced Atomization Of Liquids With Application To Ultrasonic Medical Nebulizers

Existing literature on the operation of ultrasonic vibrating mesh nebulizers does not entirely explain the principles by which these devices atomize liquid medication. Many of the studies on this topic assume a spray or extrusion mode of droplet generation, but it can be demonstrated that the high frequency vibration of these devices is sufficient to produce appropriately-sized aerosol droplets. A sufficiently small volume or thin film of liquid that is vibrated under correct conditions will produce a fountain of atomized liquid droplets which are appropriately sized for transport and deposition deep into the lungs, which is necessary for inhalation therapy. The formation of standing waves on the surface of this sort of thin film have an oscillating frequency that is roughly half the driving frequency and a wavelength that is equal to a function of the ultrasonic driving frequency, fluid density, and interfacial surface tension. The standing wavelength in particular is shown to be approximately three times the mean droplet diameter that makes up the resulting spray. Also, several studies have shown that cavitation is likely to be present in vibrating films of water which destabilize the capillary waves and may alter the overall droplet diameter distribution of the resulting fountain. This study validates these phenomena by relating existing concepts of liquid atomization to the operating parameters of known atomizing systems and the Omron Micro Air vibrating mesh nebulizer, along with numerically altering these parameters to show trends in response conditions. A CFD analysis is performed which assists in model verification and reveals that some critical configuration driving amplitude and liquid depth must be fulfilled in order for droplet kinetic energy to exceed fluid resistance energy so that the atomization process can initiate. The Omron Micro Air operates at an ultrasonic frequency of approximately 180 kHz and is able to maintain a liquid film that is the correct thickness to generate capillary waves leading to droplet ejection. The vibrating mesh component is assumed to be largely responsible for maintaining this film thickness along with acting as a sizing screen to only release droplets that are 3 µm or smaller. The exact function of the vibrating mesh is not analyzed in detail during this study, as the primary focus is to verify and identify parameters of atomization of a thin film of water under the aforementioned operating conditions