Microbubble Expansion Research Articles

Drop-on-demand cell bioprinting via Laser Induced Side Transfer (LIST)

We introduced and validated a drop-on-demand method to print cells. The method uses low energy nanosecond laser (wavelength: 532 nm) pulses to generate a transient microbubble at the distal end of a glass microcapillary supplied with bio-ink. Microbubble expansion results in the ejection of a cell-containing micro-jet perpendicular to the irradiation axis, a method we coined Laser Induced Side Transfer (LIST). We show that the size of the deposited bio-ink droplets can be adjusted between 165 and 325 µm by varying the laser energy. We studied the corresponding jet ejection dynamics and determined optimal conditions for satellite droplet-free bioprinting. We demonstrated droplet bio-printing up to a 30 Hz repetition rate, corresponding to the maximum repetition rate of the used laser. Jet ejection dynamics indicate that LIST can potentially reach 2.5 kHz. Finally, we show that LIST-printed human umbilical vein endothelial cells (HUVECs) present negligible loss of viability and maintain their abilities to migrate, proliferate and form intercellular junctions. Sample preparation is uncomplicated in LIST, while with further development bio-ink multiplexing can be attained. LIST could be widely adapted for applications requiring multiscale bioprinting capabilities, such as the development of 3D drug screening models and artificial tissues.

Moving toward Noninvasive, Focused Ultrasound Therapeutic Delivery of Drugs in the Brain: Prolonged Opening of Blood-Brain Barrier May Not Be Needed.

Moving toward Noninvasive, Focused Ultrasound Therapeutic Delivery of Drugs in the Brain: Prolonged Opening of Blood-Brain Barrier May Not Be Needed.

Enhanced microbubble contrast agent oscillation following 250\u2009kHz insonation

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

Ultrasound Cavitation/Microbubble Detection and Medical Applications

Over the past decades, different techniques have been investigated for detecting microbubbles. The purpose of this work is to review the state-of-the-art of medical microbubble detection along with therapeutic, monitoring, and diagnostic applications. The presence of microbubbles in the human body can be induced either through cavitation or exogenous introduction of bubbles. One of the effects of ultrasound is cavitation, or microbubble formation and collapse. Cavitation produces high pressures and temperatures, and microbubble expansion and then collapse close to cells can lead to cellular damage or hemorrhage in biological tissues. Cavitation is, in most cases, an undesired event in clinical diagnostic imaging. Considering that cavitation microbubble formation is largely unpredictable, ultrasound imaging may present a rare or yet unknown risk, particularly to fetuses and embryos. Although most therapeutic ultrasound modalities work based on physical and thermal effects of cavitation, the safety of treatment strongly depends on accurate knowledge of the location of the cavitation inception point. Cavitation detection is an important factor with respect to improving the safety of ultrasound imaging and therapy. It is essential to recognize the existence and location of cavitation inception points. In addition, the use of encapsulated microbubbles as contrast agents for diagnostic imaging, as vehicles for local drug or gene delivery, and as tools for microbubble and ultrasound therapy in thrombolysis has increased the demand for an accurate deep tissue microbubble detection technique. There have been many attempts to detect cavitation bubbles, but each contains its own limitations. There is no doubt that continuous discoveries and developments in microbubble detection modalities will lead to safer and more efficient therapeutic and diagnostic equipment.

Urinary stone erosion and fragmentation under low-intensity quasi-collimated ultrasound using gas-filled microbubbles with stone-targeting lipid shells

Urinary stone lithotripsy critically depends on the presence of cavitation nuclei at the stone surface. We hypothesized that introduction of stone-targeting microbubbles could increase cavitation activity at a stone surface sufficiently to allow stone erosion and fragmentation at peak negative pressures much lower than in acoustic energy-based urinary stone interventions with induced cavitation nuclei alone. Gas-filled microbubbles were produced with calcium-binding moieties incorporated into an encapsulating lipid shell. Stone surface coverage with these targeting microbubbles was found to approach an optimal (considering microbubble expansion during insonation) range of 5–15% with incubation times of three minutes or less. Using high-speed photomicroscopy, we observe bound microbubbles expanding 10- to 30-fold under insonation with quasi-collimated sources at mechanical indexes below 1.9. For observed stand-off parameters in the range of 0.2–0.6, the modeled collapse-generated shockwaves exceed 100 MPa. In swine model studies with these targeting microbubbles, stone fragmentation into passable fragments occurs with treatment times around 30 minutes, while post-treatment examination of ureters and kidneys shows no evidence of urothelium damage or renal parenchymal hemorrhage. The stone-targeting microbubbles reported on here have formed the basis for a new non-invasive urinary stone treatment which recently entered human clinical trials.

Experimental observations and numerical modeling of lipid-shell microbubbles with calcium-adhering moieties for minimally-invasive treatment of urinary stones.

A novel treatment modality incorporating calcium-adhering microbubbles has recently entered human clinical trials as a new minimally-invasive approach to treat urinary stones. In this treatment method, lipid-shell gas-core microbubbles can be introduced into the urinary tract through a catheter. Lipid moities with calcium-adherance properties incorporated into the lipid shell facilitate binding to stones. The microbubbles can be excited by an extracorporeal source of quasi-collimated ultrasound. Alternatively, the microbubbles can be excited by an intraluminal source, such as a fiber-optic laser. With either excitation technique, calcium-adhering microbubbles can significantly increase rates of erosion, pitting, and fragmentation of stones. We report here on new experiments using high-speed photography to characterize microbubble expansion and collapse. The bubble geometry observed in the experiments was used as one of the initial shapes for the numerical modeling. The modeling showed that the bubble dynamics strongly depends on bubble shape and stand-off distance. For the experimentally observed shape of microbubbles, the numerical modeling showed that the collapse of the microbubbles was associated with pressure increases of some two-to-three orders of magnitude compared to the excitation source pressures. This in-vitro study provides key insights into the use of microbubbles with calcium-adhering moieties in treatment of urinary stones.

Ultrafast frame rate microscopy of microbubble oscillations: Current studies employing the UPMC-Cam

Ultrasound-stimulated microbubbles are clinically employed for diagnostic imaging and have been shown to be a feasible therapeutic strategy for localized drug and gene delivery applications. In order to investigate microbubble oscillation dynamics, the University of Pittsburgh Medical Center has developed the UPMC-Cam—an ultrafast imaging system capable of recording 128 frames at up to 25 million frames per second, one of only two in the world currently in use. Current studies include elucidating the effect of fluid viscosity on microbubble behavior. At lower pressures (0.1–0.25 MPa), an increase in fluid viscosity from 1 to 4 cP alters the fundamental and second-harmonic oscillation amplitudes in a bubble size-dependent manner, related to resonance. At higher pressures (0.5–1.5 MPa), a significant decrease in the propensity for microbubble fragmentation was observed in the more viscid fluid environment. Additionally, studies aimed at gaining physical insights into sonoporation have been conducted in which individual bubbles lay adjacent to a cell monolayer. These studies have revealed a maximum microbubble expansion threshold above which sonoporation occurs, likely related to the associated shear stresses exerted by a microbubble on the adjacent cell membrane. The UPMC-Cam has shown to be an invaluable tool for investigating biophysical-related phenomena over a wide range of ultrasound-microbubble applications.

Experimental and theoretical studies of microbubble interaction with tissue help optimize drug delivery and imaging.

A multi‐disciplinary approach is required to engineer ultrasound contrast agents for drug delivery and imaging. These micrometer‐sized bubbles oscillate in response to acoustic energy, emitting non‐linear echoes that can be differentiated from the tissue when imaged. If large enough, the oscillations generate perturbations capable of affecting the region surrounding the bubble. Here, microbubble activity is observed in scenarios that mimic those found in vivo to provide intuition about the mechanisms for contrast‐enhanced imaging and drug delivery. High‐speed imaging of microbubble oscillation at 1 MHz within ex vivo vessels reveals that microbubble expansion is significantly reduced, and the constrained oscillation imparts a force capable of displacing the surrounding vessel wall. [Caskey et al., 122, 1191‐1200.] Bubble oscillation near a tissue‐mimicking gel boundary shows fluid jets impinging on and disrupting the gel surface at 1, 2.25, and 5 MHz. [Caskey et al., 125, EL183–EL189.] The bubble concentration and acoustic parameters associated with gel disruption and vessel displacement are examined with respect to theoretical predictions, suggesting the ratio of pressure to frequency may be a good indicator for these phenomena. Finally, we also report on extensions of this work to the interaction of targeted contrast agents with tissue.

Dynamics of acoustic droplet vaporization in gas embolotherapy

Acoustic droplet vaporization is investigated in a theoretical model. This work is motivated by gas embolotherapy, a developmental cancer treatment involving tumor infarction with gas microbubbles that are selectively formed from liquid droplets. The results indicate that there exists a threshold value for initial droplet size below which the bubble evolution is oscillatory and above which it is smooth and asymptotic, and show that the vaporization process affects the subsequent microbubble expansion. Dampening of the bubble expansion is observed for higher viscosity and surface tension, with effects more pronounced for droplet size less than 6 mum in radius.

Ultrasound-modulated fluorescence based on a fluorophore-quencher-labeled microbubble system

Ultrasound-modulated fluorescence from a fluorophore-quencher-labeled microbubble system driven by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based imaging methods are discussed in three different illumination modes of the excitation light: continuous wave (DC), frequency domain (FD), and time domain (TD). Results show that microbubble expansion opens a time period during which the quenching efficiency is dramatically reduced so that the emitted fluorescence strength and fluorophore lifetime are significantly increased. The modulation efficiency may even reach 100%. In addition, an important finding in this study is that in TD illumination mode, the modulated fluorescence photons may be temporally separated from the unmodulated photons, which makes the modulation efficiency limited only by thermal noise of the measurement system.

Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall

Many thousands of contrast ultrasound studies have been conducted in clinics around the world. In addition, the microbubbles employed in these examinations are being widely investigated to deliver drugs and genes. Here, for the first time, the oscillation of these microbubbles in small vessels is directly observed and shown to be substantially different than that predicted by previous models and imaged within large fluid volumes. Using pulsed ultrasound with a center frequency of 1 MHz and peak rarefactional pressure of 0.8 or 2.0 MPa, microbubble expansion was significantly reduced when microbubbles were constrained within small vessels in the rat cecum (p<0.05). A model for microbubble oscillation within compliant vessels is presented that accurately predicts oscillation and vessel displacement within small vessels. As a result of the decreased oscillation in small vessels, a large resting microbubble diameter resulting from agent fusion or a high mechanical index was required to bring the agent shell into contact with the endothelium. Also, contact with the endothelium was observed during asymmetrical collapse, not during expansion. These results will be used to improve the design of drug delivery techniques using microbubbles.

Microbubble Expansion in a Flexible Tube

We have utilized a computational model of the expansion of a microbubble in a liquid-filled flexible tube to investigate the potential for acoustic vaporization of perfluorocarbon droplets to damage blood vessels during a novel gas embolotherapy technique for the potential treatment of tumors. This model uses a fixed grid, multi-domain, interface tracking, direct numerical simulation method that treats all interfaces and boundaries as sharp discontinuities for high accuracy. In the current work, we examined effects of initial bubble size on the flows and wall stresses that result from droplet vaporization. The remaining dimensionless parameters that govern the system response (Reynolds, Weber, and Strouhal numbers, initial bubble pressure, and wall stiffness and tension) were selected to model an arteriole. The results for a flexible tube are significantly different from those for a rigid tube. Two major flow regimes occur due to the combined effect of bubble and tube deformation: in flow at the tube ends and out flow near the bubble surface. The flexibility of the tube largely dissipates the extreme pressure that develops in the rigid tube model. Both the magnitude and the overall expansion time of the rapidly changing pressure are greatly reduced in the flexible tube. Smaller initial bubble diameters, relative to the vessel diameter, result in lower wall stresses. This study indicates that wall flexibility can significantly influence the wall stresses that result from acoustic vaporization of intravascular perfluorocarbon droplets, and suggests that acoustic activation of droplets in larger, more flexible vessels may be less likely to damage or rupture vessels than activation in smaller and stiffer vessels.

Microbubble oscillation in tubes with diameters of 12, 25, and 195 microns

Ultrasound contrast agents are often used to measure flow rate in the microvasculature; however, the oscillation of these agents in capillary-sized tubes has not been directly observed. Here, oscillations of microbubbles are examined in microvessel phantoms with diameters similar to those of capillaries. High-speed camera images demonstrate the effects of ultrasonic pressure and tube diameter and length on microbubble expansion and fragmentation occurrence. Microbubble displacement due to radiation force is also demonstrated in a phantom microvessel.

Direct Numerical Simulations of Micro-Bubble Expansion in Gas Embolotherapy

We are currently developing a novel gas embolotherapy technique that involves the selective, acoustic vaporization of liquid perfluorocarbon droplets in or near a tumor as a possible treatment for cancer The resulting bubbles can then stick within the tumor vasculature to occlude blood flow and "starve" the tumor The potential development of high stresses during droplet vaporization is a major concern for safe implementation of this technique. No prior study, either experimentally or theoretically, addresses this important issue. In this work, the acoustic vaporization procedure of the therapy is investigated by direct numerical simulations. The nonlinear, multiphase, computational model is comprised of an ideal gas bubble surrounded by liquid inside a long tube. Convective and unsteady inertia, viscosity, and surface tension affect the bubble dynamics and are included in this model, which is solved by a novel fixed-grid, sharp-interface, moving boundary method. We assess the potential for flow-induced wall stresses to rupture the vessel or damage the endothelium during vaporization under a range of operating conditions by varying dimensionless parameters--Reynolds, Weber, and Strouhal numbers, inertial energy and initial droplet size. It is found that the wall pressure is typically highest at the start of the bubble expansion, but the maximum wall shear stress occurs at a later time. Smaller initial bubble diameters, relative to the vessel diameter, result in lower wall stresses.

Micromanipulation of endothelial cells: Ultrasound-microbubble-cell interaction

Ultrasound (US) in combination with contrast microbubbles has been shown to alter the permeability of cell membranes without affecting cell viability. This permeabilisation feature is used to design new drug delivery systems using US and contrast agents. The underlying mechanisms are still unknown. One hypothesis is that oscillating microbubbles cause cell deformation resulting in enhanced cell membrane permeability. This technical note reveals the interaction between oscillating microbubbles and endothelial cells under a microscope recorded with a fast framing camera at 10 million frames per second. A microbubble expansion of 100% resulted a 2.3-μm displacement of the cell membrane. During the insonification, changes of approximately 15% in the cross-sectional distance of the endothelial cells were observed due to microbubble vibrations. In conclusion, the use of such a camera makes it possible to reveal the mechanisms of interactions between ultrasound, microbubbles and cells. (E-mail: j.vanwamel@erasmusmc.nl)

Selective cell killing by microparticle absorption of pulsed laser radiation

The mechanism of interaction between subcellular pigment microparticles and short pulse laser radiation was investigated with nanosecond time-resolved microscopy and fluorescence microscopy. Stroboscopic illumination was used to capture images of transient events induced in cells by laser pulses. Fluorescence microscopy enabled assessment of cell damage using fluorescent probes. Short-lived intracellular cavitation bubbles were directly visualized within nanoseconds after laser irradiation. Microbubble expansion and implosion took place on the timescale of 0.1-1 /spl mu/s and were confined entirely within pigmented cells with remarkable selectivity. Cells containing particles underwent cavitation and rapidly lost viability, while adjacent cells without particles remained viable. Nonpigmented cells can be targeted by labeling them with absorbing particles. Laser-pumped microparticles provide an efficient means of selective cell targeting.