Microbubble Oscillation Research Articles

Magneto-sonothrombolysis with combination of magnetic microbubbles and nanodroplets

This paper reports a novel technique using the rotational magnetic field oscillation and low-intensity sub-megahertz ultrasound stimulation of magnetic microbubbles (MMBs) to promote the nanodroplets (NDs) phase transition and improve the permeation of NDs into the blood clot fibrin network to enhance the sonothrombolysis efficiency. In this study, the influence of different treatment methods with a combination of MMBs and NDs on the thrombolysis rate of both unretracted and retracted clots were investigated, including the stable and inertial cavitation, tPA effects, MMBs/NDs concentration ratio, sonication factors (input voltage, duty cycle) and rotational magnetic field factors (flux density, frequency). We demonstrated that tPA-mediated magneto-sonothrombolysis in combining NDs with MMBs could significantly enhance in vitro lysis of both unretracted clots (85 ± 8.3%) and retracted clots (57 ± 6.5%) in a flow model with 30 min treatment. The results showed that the combination of MMBs and NDs substantially improves in vitro lysis of blood clots with an unprecedented lysis rate.

Photoacoustic Impulse Response of Lipid-Coated Ultrasound Contrast Agents.

The utility of ultrasound imaging and therapy with microbubbles may be greatly enhanced by determining their impulse-response dynamics as a function of size and composition. Prior methods for microbubble characterization utilizing high-speed cameras, acoustic transducers and laser-based techniques typically scan a limited frequency range. Here, we report on the use of a novel photoacoustic technique to measure the impulse response of single microbubbles. Individual microbubbles are driven with a broadband photoacoustic wave generated by a nanosecond-pulse laser illuminating an optical absorber. The resulting microbubble oscillations were detected by following transmission of a second laser as it passes twice through the microbubble. The system could even resolve oscillations resulting from a single-shot. As a proof-of-concept study, the size-dependent, linear impulse response of lipid-coated microbubbles was characterized using this technique. This unique method of microbubble characterization with exceptional spatiotemporal resolution opens new avenues for capturing and analyzing microbubble system dynamics.

Theoretical analysis on thermal effect of weakly nonlinear propagation of focused ultrasound toward medical applications

Toward medical applications such as a tumor ablation therapy by High-Intensity Focused Ultrasound (HIFU), weakly nonlinear propagation of quasi-planar focused ultrasound in a liquid containing many spherical microbubbles is theoretically investigated, especially focusing on thermal effects of microbubble oscillations. We shall derive a Khokhlov–Zabolotskaya–Kuznetsov (KZK) type equation incorporating the thermal effects of the microbubble oscillations by introducing the energy equation proposed by Prosperetti [J. Fluid Mech. (1991)] into the set of basic equations based on mixture and two-fluid models. Moreover, the phase difference between the temperature gradient and the average temperature inside bubble is considered in the energy equation. As a result, we found that (i) the thermal conduction of gas inside bubbles strongly affects the dissipation of ultrasound and (ii) the phase difference affects the advection of ultrasound.

Oscillatory behavior of microbubbles impacts efficacy of cellular drug delivery

Drug-loaded microbubbles have been proven to be an effective strategy for non-invasive and local drug delivery when combined with ultrasound excitation for targeted drug release. Inertial cavitation is speculated to be a major mechanism for releasing drugs from drug-loaded microbubbles, but it results in lethal cellular pore damage that greatly limits its application. Thus, we investigated the cellular vesicle attachment and uptake to evaluate the efficiency of drug delivery by modulating the behaviors of targeted microbubble oscillation. The efficiency of vesicle attachment on the targeted cell membrane was 36.5 ± 15.9% and 3.8 ± 2.3% under stable and inertial cavitation, respectively. Further, stable cavitation enhanced cell permeability (26.8 ± 3.2%), maintained cell viability (90.8 ± 2.1%), and showed 7.9 ± 1.9-fold enhancement of in vivo vesicle release on tumor vessels. Therefore, our results reveal the ability to improve drug delivery via stable cavitation induced by targeted microbubbles. We propose that this strategy might be suitable for tissue repair or neuromodulation.

Vibrational behavior of coated microbubble in finite tube under magneto-acoustic composite field

The dynamic behavior of coated microbubble in a magneto-acoustic field is very significant for its application to therapy. In this paper, the radial vibration equation of microbubble is derived by placing the coated-microbubbles in a tube filled with magnetic fluid and considering the magnetic pressure on the magnetic fluid under the magneto-acoustic field. The dynamic equation is nondimensionalized by using characteristic quantities such as Weber number and Reynolds number. The effects of magnetic-acoustic field parameters and magnetofluid characteristics on the vibration behavior of the vibration system are analyzed by the Runge-Kutta method. The results show that the magnetic field can prevent the collapse and make bubble oscillate stably. When the acoustic field is constant, the magnetic field can stabilize the oscillation of the microbubble and increase the equilibrium radius of the oscillating microbubble. The stronger the magnetic field is, the more obvious the influence of magnetofluid magnetisability <i>χ</i><sub>m</sub> on the vibration of the microbubble is and the stronger the nonlinear of the transient response of the microbubble is when the magnetic field is larger. In addition, the larger acoustic field parameters will enhance the response of oscillating microbubble to magnetic field. The larger the magnetic field is, the weaker the influence of acoustic parameters on the oscillations of microbubble is. Also, the transient response of microbubble is obviously nonlinear, but the steady-state response keeps the reciprocating oscillation with small amplitude. It can be seen that the adjusting of the magneto-acoustic field is beneficial to realizing the stable oscillation of microbubble in the blood vessel and avoiding collapse.

Effects of contrast-enhanced ultrasound treatment on neoadjuvant chemotherapy in breast cancer.

Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer.Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC).Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors.Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation.

Phospholipid bilayer responses to ultrasound-induced microbubble cavitation phenomena

Abstract Using liposomal bilayers as a model for bacterial cell membranes, this study investigates the extents to which high-frequency (1.0 and 3.3 MHz) ultrasound-induced microbubble cavitation phenomena are effective for bacterial inactivation. Forster Resonance Energy Transfer (FRET), acoustic scattering and a mathematical model were used to quantify the proximity of microbubbles to liposomes, establish if liposomes affect microbubble cavitation and propose bilayer alterations' mechanisms. Addition of a positive charge on microbubbles increased their proximity to liposomes through electrostatic attraction, however, dampened microbubble's oscillation compared to freely-floating microbubbles. Alleviation of energy transfer due to phospholipid mixing between microbubbles and liposomes established that close-proximity is necessary for bilayer alterations. Approximately 19% mixing of phospholipids was observed at 3.3 MHz due to microstreaming from stable cavitation, while inertial cavitation at 1.0 MHz increased this mixing to 50%. This method can damage bacterial membrane; but, whether bacterial defense mechanisms can attenuate this effect remains to be understood.

Nonlinear dynamics of a shell encapsulated microbubble near solid boundary in an ultrasonic field

Microbubbles have the potential to be used for diagnostic imaging and therapeutic delivery. However, the transition from microbubbles currently being used as ultrasound contrast agents to achieve its’ potentials in the biomedical field requires more in depth understanding. Of particular importance is the influence of microbubble encapsulation of a microbubble near a vessel wall on the dynamical behaviour as it stabilizes the bubble. However, many bubble studies do not consider shell encapsulation in their studies. In this work, the dynamics of an encapsulated microbubble near a boundary was studied by numerically solving the governing equations for microbubble oscillation. In order to elucidate the effects of a boundary to the non-linear microbubble oscillation the separation distances between microbubble will be varied along with the acoustic driving. The complex nonlinear vibration response was studied in terms of bifurcation diagrams and the maximum radial expansion. It was found that the increase in distance between the boundary and the encapsulated bubble will increase the oscillation amplitude. When the value of pressure amplitude increased the single bubble is more likely to exhibit the chaotic behaviour and maximum radius also increase as the inter wall-bubble distance is gradually increased. While, with higher driving frequency the maximum radial expansion decreases and suppress the chaotic behaviour.

Numerical study on a polymer-shelled microbubble submerged in soft tissue

Ultrasound contrast agents have been recently utilized in therapeutical implementations for targeted delivery of pharmaceutical substances. Radial pulsations of the encapsulated microbubbles under the action of an ultrasound field are complex and high nonlinear, particularly for drug and gene delivery applications with high acoustic pressure amplitudes. The dynamics of a polymer-shelled agent are studied through applying the method of chaos physics whereas the effects of the outer medium compressibility and the shell were considered. The stability of the ultrasound contrast agent is examined by plotting the bifurcation diagrams, Lyapunov exponent, and time series over a wide range of variations of influential parameters. The findings of the study indicate that by tuning the shear modulus of surrounding medium and shell viscosity, the radial oscillations of microbubble cluster undergoes a chaotic unstable region as the amplitude and frequency of ultrasonic pulse are increased mainly due to the period doubling phenomenon. Furthermore, influences of various parameters which present a comprehensive view of the radial oscillations of the microbubble are quantitatively discussed with clear descriptions of the stable and unstable regions of the microbubble oscillations for typical therapeutic ultrasound pulses.

Targeting and deep-penetrating delivery strategy for stented coronary artery by magnetic guidance and ultrasound stimulation.

Stent placement is an effective treatment for atherosclerosis, but is suffered from in-stent restenosis (ISR) caused by stent mechanical damage. Conventional ISR treatment such as drug-eluting stents (DES) is challenged by the low therapeutic efficacy and severe complications, unchangeable drug dosage for individuals, and limited drug penetration in the vascular tissue. We hypothesize that magnetic targeting and deep-penetrating delivery strategy by magnetic guidance and ultrasound stimulation might be an effective approach for ISR treatment. In the present study, antiproliferative drug (paclitaxel, PTX) loaded poly (lactide-co-glycolide) (PLGA) nanoparticles (PLGA-PTX) were embedded within the shells of the magnetic nanoparticle coated microbubbles (MMB-PLGA-PTX). Once being targeted to the stent under a magnetic field, a low intensity focused ultrasound (LIFU) is applied to activate stable microbubble oscillations, thereby triggering the release of PLGA-PTX. The generated mechanical force and microstreaming facilitate the penetration of released PLGA-PTX into the thickened vascular tissue and enhance their internalization by smooth muscle cells (SMCs), thereby reducing the clearance by blood flow. In an ex vivo experiment, magnetic targeting improved the accumulation amount of MMB-PLGA-PTX by 10 folds, while the LIFU facilitated the penetration of released PLGA-PTX into the tunica media region of the porcine coronary artery, resulting in prolonged retention time at the stented vascular tissue. With the combination effects, this strategy holds great promise in the precision delivery of antiproliferative drugs to the stented vascular tissue for ISR treatment.

Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

High-Resolution Imaging of Intracellular Calcium Fluctuations Caused by Oscillating Microbubbles

Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbubble–cell interaction remains poorly understood. Because intracellular calcium (Cai2+) is a key cellular regulator, unraveling the Cai2+ fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Cai2+ fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Cai2+ uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cell–cell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 μm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Cai2+ fluctuations, providing new insight into the microbubble–cell interaction to aid clinical translation.

Opening of endothelial cell–cell contacts due to sonoporation

Ultrasound insonification of microbubbles can locally increase vascular permeability to enhance drug delivery. To control and optimize the therapeutic potential, we need to better understand the underlying biological mechanisms of the drug delivery pathways. The aim of this in vitro study was to elucidate the microbubble-endothelial cell interaction using the Brandaris 128 ultra-high-speed camera (up to 25 Mfps) coupled to a custom-built Nikon confocal microscope, to visualize both microbubble oscillation and the cellular response. Sonoporation and opening of cell-cell contacts by single αVβ3-targeted microbubbles (n = 152) was monitored up to 4 min after ultrasound insonification (2 MHz, 100–400 kPa, 10 cycles). Sonoporation occurred when microbubble excursion amplitudes exceeded 0.7 μm. Quantification of the influx of the fluorescent model drug propidium iodide upon sonoporation showed that the size of the created pore increased for larger microbubble excursion amplitudes. Microbubble-mediated opening of cell-cell contacts occurred as a cellular response upon sonoporation and did not correlate with the microbubble excursion amplitude itself. The initial integrity of the cell-cell contacts affected the susceptibly to drug delivery, since cell-cell contacts opened more often when cells were only partially attached to their neighbors (48%) than when fully attached (14%). The drug delivery outcomes were independent of nonlinear microbubble behavior, microbubble location, and cell size. In conclusion, by studying the microbubble–cell interaction at nanosecond and nanometer resolution the relationship between drug delivery pathways and their underlying mechanisms was further unraveled. These novel insights will aid the development of safe and efficient microbubble-mediated drug delivery.

Effects of varying ultrasound parameters on the oscillation of a microbubble near a boundary

Microbubbles are currently used as ultrasound contrast agents. However, microbubbles have the potential to be used in diagnostic imaging and therapeutic applications. Most numerical work on simulating microbubble oscillation involves modelling microbubbles in infinite medium whereas clinical trials and experiments involve microbubbles near a boundary such as an endothelium and blood vessels. This study will extend previous research by investigating the effects of varying the ultrasound parameters on a microbubble near a boundary. The microbubble oscillation was found to become increasingly irregular as the driving pressure amplitude is increased. The maximum amplitude of oscillation was found to decrease with increasing ultrasound frequency.

Changes in microbubble dynamics upon adhesion to a solid surface.

The interaction between an acoustically driven microbubble and a surface is of interest for a variety of applications, such as ultrasound imaging and therapy. Prior investigations have mainly focused on acoustic effects of a rigid boundary, where it was generally observed that the wall increases inertia and reduces the microbubble resonance frequency. Here we investigate the response of a lipid-coated microbubble adherent to a rigid wall. Firm adhesion between the microbubble and a glass surface was achieved through either specific (biotin/avidin) or nonspecific (lipid/glass) interactions. Total internal reflection fluorescence microscopy was used to verify conditions leading to either adhesion or non-adhesion of the bubble to a glass or rigid polymer surface. Individual microbubbles were driven acoustically to sub-nanometer-scale radial oscillations using a photoacoustic technique. Remarkably, adherent microbubbles were shown to have a higher resonance frequency than non-adherent microbubbles resting against the wall. Analysis of the resonance curves indicates that adhesion stiffens the bubble by an apparent increase in the shell elasticity term and decrease in the shell viscosity. Based on these results, we conclude that surface adhesion is dominant over acoustic effects for low-amplitude microbubble oscillations.

Lipid-based microbubbles and ultrasound for therapeutic application.

Microbubbles with diagnostic ultrasound have had a long history of use in the medical field. In recent years, the therapeutic application of the combination of microbubbles and ultrasound, called sonoporation, has received increased attention as microbubble oscillation or collapse close to various barriers in the body was recognized to potentially open those barriers, increasing drug transport across them. In this review, we aimed to describe the development of lipid-stabilized microbubbles equipped with functions, such as long circulation and drug loading, and the therapeutic application of sonoporation for tumor-targeted therapy, brain-targeted therapy, and immunotherapy. We also attempted to discuss the current status of the field and potential future developments.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed “sonoprinting”. However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

Dynamics of an ultrasound contrast agent microbubble near spherical boundary in ultrasound field**Project supported by the National Natural Science Foundation of China (Grant Nos. 11774088 and 11474090), the Hunan-Provincial Natural Science Foundation of China (Grant No. 13JJ3076), and the Science Research Program of Education Department of Hunan Province of China (Grant No. 14A127).

The goal of this article is to establish the conditions of excitation where one has to deal with ultrasound contrast agent (UCA) microbubbles pulsating near biological tissues with spherical boundary in ultrasound field for targeted drug delivery and cavitation-enhanced thrombolysis, etc., and contributes to understanding of mechanisms at play in such an interaction. A modified model is presented for describing microbubble dynamics near a spherical boundary (including convex boundary and concave boundary) with an arbitrary-sized aperture angle. The novelty of the model is such that an oscillating microbubble is influenced by an additional pressure produced by the sound reflection from the boundary wall. It is found that the amplitude of microbubble oscillation is positively correlated to the curve radius of the wall and negatively correlated to the aperture angle of the wall and the sound reflection coefficient. Moreover, the natural frequency of the microbubble oscillation for such a compliable wall increases with the wall compliance, but decreases with the reduction of the wall size, indicating distinct increase of the natural frequency compared to a common rigid wall. The proposed model may allow obtaining accurate information on the radiation force and signals that may be used to advantage in related as drug delivery and contrast agent imaging.

Microbubble based sonoporation — from the basics into clinical implications

Sonoporation is a rapidly developing novel technique serving for drug delivery and non-viral gene therapy. It is based on the interaction between microbubbles located in the surrounding of a cell and its membrane. The interaction is obtained by excitation of microbubbles with ultrasounds. This leads to reversible cell membrane poration. Depending on the intensity of ultrasounds, structure of microbubbles used in an experiment and different environmental factors, microbubbles can interact in two manners. First, in lower ultrasound intensities, stable cavitation – regular microbubbles oscillations due to changes in the environment pressure. Microbubbles have to be very close to a cell membrane, therefore, they are usually targeted to an antigen located on the cell membrane by antibodies. Consequently, microbubbles push and pull on the cell membrane and create microstreaming around it causing its disruption. Second, inertial cavitation, where in contrary to the previous one, oscillations cause rapid collapse of microbubbles, which creates shock waves and microjets for the same purpose. No matter in which manner prorated, cells tend to reseal their disrupted cell membrane. Ca2+ ions play a crucial role in the process as well as endo exocytosis. Sonoporation has proved to be an effective modality against different diseases, including variety of cancer types in of both laboratory and clinical studies.

Numerical study on formation of an acoustic soliton in bubbly liquids based on weakly nonlinear wave equation

Oscillation of microbubbles in bubbly liquids induces a dispersion effect of waves into weakly nonlinear pressure waves, and its propagation process is described by a KdV-Burgers (KdVB) equation for a long wave case. We numerically predict an evolution of waveform by solving the KdVB equation derived by Kanagawa et al. (2010, 2011) via a finite difference method. As a result, (i) an initial Gaussian waveform is distorted due to a nonlinear effect, and a steepening of waveform is observed on about 20 000 period; (ii) the dispersion effect is observed at a steep part of waveform, and its part becomes sharp as the initial void fraction increases; (iii) the initial waveform change into an attenuated soliton on about 100 000 period due to a dissipation effect, and the attenuation of soliton is noteworthy as the initial void fraction decreases; (iv) balance of the nonlinear, dispersion, and dissipation effects thereby forms an attenuated soliton, and its amplitude strongly depends on the value of the initial void fraction. Furthermore, the period of soliton formation depends on the type of initial waveform. Although the nonlinear, dissipation, and dispersion effects were qualitatively balanced in the KdVB equation, we conclude that these effects independently appear in the sound field.