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

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

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

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 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