Institute of Fundamental Technological Research

New ultrasound contrast agents that incorporate a therapeutic compound have become of interest. Such an ultrasound contrast agent particle might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load could be released with the assistance of ultrasound. Generally, an increase in shell thickness increases the acoustic amplitude needed to disrupt a bubble. High acoustic amplitudes, however, have been associated with unwanted effects on cells. It would be interesting to incorporate a droplet containing drugs or genes inside a microbubble carrier. A liquid core surrounded by a gas encapsulation has been referred to as antibubble. In this paper, the creation of an antibubble with the aid of ultrasound has been demonstrated with high-speed photography.

Antibubble, Ultrasound contrast agent, Drug delivery, High-speed photography

Nitric oxide (NO) has been implicated in smooth muscle relaxation. Its use has been widespread in cardiology. Due to the effective scavenging of NO by hemoglobin, however, the drug has to be applied locally or in large quantities, to have the effect desired. We propose the use of encapsulated microbubbles that act as a vehicle to carry the gas to a region of interest. By applying a burst of high-amplitude ultrasound, the shell encapsulating the gas can be cracked. Consequently, the gas is released upon which its dissolution and diffusion begins. This process is generally referred to as (ultra)sonic cracking.
To test if the quantities of released gas are high enough to allow for NO-delivery in small vessels (ø < 200 lm), we analyzed high-speed optical recordings of insonified stiff-shelled microbubbles. These microbubbles were subjected to ultrasonic cracking using 0.5 or 1.7 MHz ultrasound with mechanical index MI > 0.6. The mean quantity released from a single microbubble is 1.7 fmol. This is already more than the NO production of a 1 mm long vessel with a 50 lm diameter during 100 ms. However, we simulated that the dissolution time of typical released NO microbubbles is equal to the half-life time of NO in whole blood due to scavenging by hemoglobin (1.8 ms), but much smaller than the extravascular half-life time of NO (>90 ms).
We conclude that ultrasonic cracking can only be a successful means for nitric oxide delivery, if the gas is released in or near the red blood cell-free plasma next to the endothelium. A complicating factor in the in vivo situation is the variation in blood pressure. Although our simulations and acoustic measurements demonstrate that the dissolution speed of free gas increases with the hydrostatic pressure, the in vitro acoustic amplitudes suggest that the number of released microbubbles decreases at higher hydrostatic pressures. This indicates that ultrasonic cracking mostly occurs during the expansion phase.

Nitric oxide, Sonic cracking

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical microbubbles have been studied with the aid of high-speed photography. We filmed medical microbubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles with thin lipid shells have been observed to act as microsyringes during one single ultrasonic cycle. This jetting phenomenon presumably causes sonoporation. Furthermore, we observed that the gas content can be forced out of albumin-encapsulated microbubbles. These free bubbles have been observed to jet, too. It is concluded that microbubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by diagnostic ultra- sound. This opens up a whole new area of potential applications of diagnostic ultrasound related to targeted imaging and therapeutic delivery of drugs such as nitric oxide.

High-speed photography, Ultrasound contrast agent, Therapeutic microbubbles

We investigated gas release from two hard- shelled ultrasound contrast agents by subjecting them to high-mechanical index (MI) ultrasound and simultaneously capturing high-speed photographs. At an insonifying frequency of 1.7 MHz, a larger percentage of contrast bubbles is seen to crack than at 0.5 MHz. Most of the released gas bubbles have equilibrium diameters between 1.25 and 1.75 m. Their disappearance was observed optically. Free gas bubbles have equilibrium diameters smaller than the bubbles from which they have been released. Coalescence may account for the long dissolution times acoustically observed and published in previous studies. After sonic cracking, the cracked bubbles stay acoustically active.

Sonic cracking

Nanoshelled microbubbles are suitable markers for perfused areas in ultrasonic imaging, and have potential applications in therapy. With radii up to 5 microns, their resonance frequencies are in the lower megahertz range. We explored the physics of nanoshelled microbubbles, with special attention to the influence of the nanoshell on the oscillation offset with respect to the driving phase. Microbubbles above resonance size oscillate π rad out of phase with respect to microbubbles under resonance size. As the damping becomes less, this transition in offset becomes more abrupt. Therefore, the damping due to the friction of the nanoshell can be derived from this abruptness. We support our results with some high-speed optical observations of oscillating microbubbles in an ultrasonic field.

Purpose:
Ultrasound contrast agents consist of bubbles in the micrometer range encapsulated by nanoshells. These medical microbubbles oscillate linearly upon insonification at low acoustic amplitudes, but demonstrate highly nonlinear, destructive behavior at relatively high acoustic amplitudes. Therefore, medical microbubbles have been investigated for their potential applications in local drug and gene delivery. We used fast-framing photography at more than a million frames per second to investigate medical microbubbles in a diagnostic ultrasonic field. In this presentation, we give an overview of the physical mechanisms of medical microbubble destruction.

Methods and Materials:
Three ultrasound contrast agents were studied with high-speed photography during insonification. The agents were inserted through a cellulose capillary with a diameter of 0.2mm. The capillary was positioned below a microscope whose optical focus coincided with the ultrasonic focus. We captured images of insonified medical bubbles at higher frame rates than the ultrasonic frequency transmitted (typically 0.5MHz). The acoustic amplitudes corresponded to mechanical indices between 0.03 and 0.8. To compare theory and experiments, we simulated insonified medical microbubble behavior, based on the behavior of large, unencapsulated bubbles in an acoustic field.

Results:
At low acoustic amplitudes (mechanical index <0.1) bubbles pulsate moderately, as predicted from theory. At high amplitudes (mechanical index >0.6) their elongated expansion phase is followed by a violent collapse. Microbubbles have been observed to coalesce (merge), fragment, crack, and jet (act as a microsyringe) during one single ultrasonic cycle. From our observations of jetting through medical bubbles, we computed that the pressure at the tip of the jet is high enough to penetrate any human cell. One image sequence reveals the temporary formation of a liquid drop inside a microbubble.

Conclusions:
Medical microbubble oscillation and translation can be modeled using large, unencapsulated bubble theory. The number of fragments generated by untrasound-induced microbubble break-up has been related to the energy absorbed by the microbubble. Medical bubbles might be used as vehicles that carry a drug to a region of interest, where the release can be controlled with ultrasound. Liquid jets may act as microsyringes, injecting a drug into target tissue. Microbubble phenomena also have potential applications in imaging and noninvasive pressure measurements.

Microbubble, Ultrasound

We studied the interaction of ultrasound contrast agent bubbles coated with a layer of lipids, driven by 0.5 MHz ultrasound. High-speed photography on the submicrosecond timescale reveals that some bubbles bounce off each other, while others show very fast coalescence during bubble expansion. This fast coalescence cannot be explained by dissipation-limited film drainage rates. We conclude that the lipid shell ruptures upon expansion, exposing clean free bubble interfaces that support plug flow profiles in the film and inertia-limited drainage whose time scales match those of the observed coalescence.

Microbubble coalescence, Ultrasound contrast agent, Film drainage, High-speed photography

When encapsulated microbubbles are subjected to high-amplitude ultrasound, the following phenomena have been reported: oscillation, translation, coalescence, fragmentation, sonic cracking and jetting. In this paper, we explain these phenomena, based on theories that were validated for relatively big, free (not encapsulated) gas bubbles. These theories are compared with high-speed optical observations of insonified contrast agent microbubbles. Furthermore, the potential clinical applications of the bubble-ultrasound interaction are explored. We conclude that most of the results obtained are consistent with free gas bubble theory. Similar to cavitation theory, the number of fragments after bubble fission is in agreement with the dominant spherical harmonic oscillation mode. Remarkable are our observations of jetting through contrast agent microbubbles. The pressure at the tip of a jet is high enough to penetrate any human cell. Hence, liquid jets may act as remote-controlled microsyringes, delivering a drug to a region-of-interest. Encapsulated microbubbles have (potential) clinical applications in both diagnostics and therapeutics.

Encapsulated microbubbles, Ultrasound contrast agent, Radiation forces, Coalescence, Fragmentation, Jets

This paper describes a noninvasive method to measure local hydrostatic pressures in fluid filled cavities. The method is based on the disappearance time of a gas bubble, as the disappearance time is related to the hydrostatic pressure. When a bubble shrinks, its response to ultrasound changes. From this response, the disappearance time, and with it the hydrostatic pressure, can be determined.
We investigated the applicability of the gases Ar, C3F8, Kr, N2, Ne, and SF6, based on their diffusive properties. For pressure measurements with a limited duration, e.g. 150 ms, Kr and Ar bubbles are most suitable, since they are most sensitive to pressure change. If there is also a limitation to bubble size, e.g. a maximum diameter of 6 lm, SF6 is most suitable.
We present improvements of a method that correlates the duration of the decay of the fundamental ultrasound response to the hydrostatic overpressure. We propose to correlate the duration until subharmonic occurrence in combination with its decay, to hydrostatic overpressure, since the subharmonic decays more rapidly than the fundamental response. For a dissolving Ar gas bubble with an initial diameter of 14 lm, the overpressure can be determined 4 times as precise from the decay of the subharmonic response as from the decay of the fundamental response. Overpressures as small as 11 mmHg may be discriminated with this method.

Noninvasive pressure measurement, Blood pressure, Microbubble, Sonic cracking

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical bubbles have been studied with the aid of high-speed photography. We filmed medical bubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles have - among others - been observed to fragment and jet during one single ultrasonic cycle. Gas was released from encapsulated microbubbles. It is concluded that bubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by ultrasound whose amplitudes are still in the diagnostic range.

Oscillating bubbles, Targeted drug delivery, Micro-injection needles

Ultrasound contrast agents (UCAs) are used in a clinical setting to enhance the backscattered signal from the blood pool to estimate perfusion and blood flow. The UCAs consist of encapsulated microbubbles, measuring 1–10 m in diameter. Acoustic characterization of UCAs is generally carried out from an ensemble of bubbles. The measured signal is a complicated summation of all signals from the individual microbubbles. Hence, characterization of a single bubble from acoustic measurements is complex.
In this study, 583 optical observations of freely flowing, oscillating, individual microbubbles from an experimental UCA were analyzed. The excursions during ultra- sound exposure were observed through a microscope. Images were recorded with a high frame rate camera operating at 3 MHz. Microbubbles on these images were measured off-line, and maximal excursions were determined. A technique is described to determine the diameters of the bubbles observed. We compared the maximal excursions of microbubbles of the same initial size in an ultrasound field with a 500 kHz center frequency at acoustic amplitudes ranging from 0.06 MPa to 0.85 MPa.
It was concluded that maximal excursions of identical bubbles can differ by 150% at low acoustic pressures (mechanical index or MI 0.2). At a high acoustic pressure (MI = 1.2) an image sequence was recorded on which a bubble collapsed, but an apparently identical bubble survived.

The cover page shows a sequence of microscopic image frames of a freely flowing contrast agent microbubble. The frames were taken during one cycle of ultrasound insonification, with a center frequency of 500 kHz. The peak negative acoustic pressure at the region of interest was 0.85 MPa. Each frame corresponds to a 45 x 27 μm2 area. The exposure time of each frame was 10 ns. Interframe times were 330 ns, except for the time between frames e and f, which was 660 ns. The sequence shows a growing gas encapsulated microbubble of 5.3 μm (a) and 17.6 μm (b), and its maximal growth of 22.9 μm (c). After shrinking to 20.2 μm (d), it ruptured (e). The microbubble had been pushed to the lower left side of the frame, apparently by water that was propelled into the microbubble. A subframe shows the negative of the region of interest. Finally, the deformed mcrobubble re-occurred as an assymetric shape (f). Understanding of microbubble-rupturing behavior is neccessary for developments in medical release burst imaging and ultra- sound-guided drug delivery. This work has been supported by the Technology Foundation STW (RKG.5104) and the Interuniversity Cardiology Institute of The Netherlands.

Ultrasound contrast agents consist of small encapsulated bubbles with diameters below 10 μ m. The encapsulation influences the behavior of these microbubbles when they are insonified by ultrasound. The highly nonlinear behavior of ultrasound contrast agents at relatively high acoustic amplitudes (mechanical index>0.6) has been attributed to nonlinear bubble oscillations and to bubble destruction. For microbubbles with a thin, highly elastic nanoshell, it has been demonstrated that the presence of the nanoshell becomes negligible at high insonifying amplitudes. From our simulations it follows that the Blake critical radius is not valid for microbubble fragmentation. The low maximal excursion observed and simulated for a thick, stiff-shelled microbubble is in agreement with previous acoustic analyses. The ultrasound-induced gas release from stiff-shelled bubbles has been reported. However, we also observed gas release from microbubbles with a thin, elastic shell.

Ultrasound contrast agent, Encapsulated microbubble, Nanoshell

Ultrasound contrast agents have been investigated for their potential applications in local drug and gene delivery. A microbubble might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load has to be released with the assistance of ultrasound. We investigate the suitability of antibubbles for ultrasound-assisted local delivery. As opposed to bubbles, antibubbles consist of a liquid core surrounded by a gas encapsulation. Incorporating a liquid drop containing drugs or genes inside an ultrasound contrast agent microbubble, however, is technically challenging.
An ultrasound-insonified microbubble generates a pressure field that is inversely proportional to the distance from the microbubble. Therefore, an oscillating contrast agent microbubble may create a surface instability with a relatively big bubble at a short distance. For big enough instabilities, a drop may be formed inside the big bubble.
Three different contrast agents were subjected to 0.5 MHz ultrasound, with mechanical indices >0.6. The contrast agents were inserted through an artificial capillary which led through the acoustic focus of the transducer. High-speed photographs were captured at a speed of 3 million frames per second and higher. We observed that ultrasound contrast microbubbles below resonance size may create visible surface instabilities with bubbles above resonance size. With an albumin-shelled contrast agent, we induced a surface instability that was big enough to create an antibubble inside a free (unencapsulated) gas bubble with an 8 micron diameter. The surface instability has been attributed to the presence of a contrast microbubble with a 3 micron diameter. This instability has the form of a re-entrant jet protruding into the gas bubble. The inward protrusion grew and subsequently drained, leaving a droplet with a five micron diameter inside the bubble. In a subsequent recording after 100 ms, only the gas bubble could be detected. Thus, the life- time of the antibubble was less then 100 ms. The presence of a surfactant on the interfaces might lead to an improved stability of an antibubble.

Antibubble, Ultrasound

The disruption of contrast agent microbubbles has been implicated in novel techniques for high-MI imaging and local drug delivery. At MI>0.6, microbubble fragmentation has been observed with thin-shelled agent (≈10nm), and shell rupture with thick-shelled agent (≈250nm). To predict the disruption of these nanoshelled microbubbles, destruction thresholds have been under investigation. In several studies, the Blake threshold pressure was associated with microbubble destruction. The Blake threshold pressure is the peak rarefactional acoustic pressure at which the critical Blake radius is reached, approximately twice the equilibrium radius, above which a bubble behaves like an inertial cavity. We studied the acoustic pressures at which a thin-shelled microbubble fragments and those at which a thick-shelled microbubble cracks. More specifically, we investigated the validity of the Blake threshold for these phenomena. The oscillating and fragmenting behavior of microbubbles with a 10nm shell was simulated at a driving frequency of 0.5–2 MHz, using a modified Rayleigh-Plesset equation and assuming that fragmentation occurs when the kinetic energy of the microbubble surpasses the instantaneous bubble surface energy. For microbubbles with radii between 1 and 6μm, the fragmentation thresholds lie between 20 and 200 kPa. Generally, the critical radius is much smaller than twice the equilibrium radius. The moment of break-up during the collapse phase is in agreement with high- speed optical observations that were presented previously.
Furthermore, the shell rupture behavior of microbubbles with a thick shell was analyzed for quasistatic pressure changes (relatively low ultrasonic frequencies), assuming that the shell obeys Hooke’s Law. The rupture threshold pressure of −80 kPa had been determined from acoustical data. For shells with the typical Young’s modulus 2MPa and Poisson ratio 0.5, this is in agreement with the observation that the maximal excursion upon rupture of such bubbles is smaller that 0.3μm.
In conclusion, we may state that the Blake threshold is neither a good estimator for the fragmentation, nor for the rupture of contrast agent microbubbles.

Shell rupture, Fragmentation threshold, Blake threshold

When gas bubbles collide, the following stages of bubble coalescence have been reported: flattening of the opposing bubble surfaces prior to contact, drainage of the interposed liquid film toward a critical minimal thickness, rupture of the liquid film, and formation of a single bubble. During insonification, expanding contrast agent microbubbles may collide with each other, resulting in coalescence or bounce.
In this study, we investigate the validity of the film drainage formalism for expanding free bubbles, by subjecting rigid-shelled contrast agent microbubbles to ultrasound, in order to release gas, and photograph the coalescence of these free gas bubbles. As with colliding bubbles, bubble surface flattening is related to the Weber number. Only inertial film drainage between free interfaces explains the observed coalescence times. In accordance with theory, smaller bubble fragments coalesce on very small time scales, while larger bubbles bounce off each other.

Understanding the mechanisms of microbubble destruction is needed for the development of ultrasound guided drug and gene delivery methods and for the improvement of diagnostic ultrasonic contrast agent (UCA) detection methods. We performed 482 experiments on the coalescence and collapse mechanisms of a soft- shelled and a hard-shelled contrast agent, by subjecting an experimental lipid-shelled UCA and the hard-shelled UCA QuantisonTM to 500 kHz, high- pressured ultrasound (MI≈1.0), and recording microscopic images of these events with a fast- framing camera. Results showed that bubble fragmentation into smaller bubbles is the primary mechanism for lipid-shelled contrast microbubble destruction during the first cycles after ultrasound arrival. In 28% of our experimental events with a lipid-shelled UCA, we observed bubble coalescence. The coalescence mechanism was observed to be analog to the process desribed for larger gas bubbles. Repetitive coalescence and fragmentation was clearly recorded with a fast-framing camera. We also demonstrated the formation and collapse of large lipid-shelled bubbles and bubble clusters. Furthermore we showed that sonic cracking is feasible for the hard-shelled contrast agent QuantisonTM.

In this study we analyze the behavior of individual experimental ultrasonic contrast bubbles, insonofied by 500 kHz ultrasound, at acoustic pressures between 0.06 and 0.66 MPa. The oscillations were observed under a microscope with a fast framing camera.
It is concluded that apparently identical bubbles can expand to different maximal diameters.