Figure 1. Schematic of an echogenic liposome

The novelty of this strategy lies in providing doctors with the capability to control the amount of drug released and delivered at the target site, all tailored to the patients needs. This can be achieved by manipulating the way ultrasound pulses interact with the liposomes. The nature of this interaction is governed by the gas within the liposomes, which makes them echogenic, meaning that they are highlighted on an ultrasound image.

In previous publications, we have shown that a clot-busting drug can be encapsulated in echogenic liposomes and released using diagnostic Doppler ultrasound provided by a clinical scanner. This release was found to be associated with the disappearance of contrast on ultrasound images.

In this work, the sensitivity of the echogenic liposomes to a variety of types of ultrasound was studied. Depending on the amplitude of the Doppler ultrasound pulses, the liposomes can respond in three possible ways: the intact vesicles may be pushed away, the vesicles may gently ring (stable cavitation), or the vesicles can be violently popped (inertial cavitation).

Both of the cavitation reactions have been found to enhance delivery of drugs and genes across cell walls and tissues. Stable cavitation seems to massage the cells, coaxing them to open up temporarily and thus allow drug delivery without destroying the cells. Inertial cavitation, on the other hand, seems to have the ability to poke a more permanent hole in the cell, which can potentially kill the cell.

In order to achieve the desired therapeutic effect and to examine the possibility of negative bioeffects, we needed to find the sweet spot, or pressure amplitude at which the ultrasound would gently shake the bubbles but not violently pop them. Fortunately, each type of cavitation results in a characteristic echo. Thus by recording the echoes, in addition to simultaneously capturing standard ultrasound images, we can begin to understand how the imaging of the echogenic liposomes may be coupled to their biological effect.

The experiments were performed by infusing the liposomes into a flow system consisting of a pump and tubing that mimic blood flow in an artery. The liposomes were hit with ultrasound pulses, similar to those that doctors use to listen to the heartbeat of a fetus. A separate ultrasound listening device recorded the echoes. Simultaneously, we recorded the loss of contrast in an ultrasound image that resulted from the ultrasound interacting with the liposomes.

 

Figure 2. Image of echogenic liposomes flowing in a latex tube hit by ultrasound Doppler pulses at the center (indicated by white vertical bars). Flow is from right to left. Disappearance of contrast of echogenic liposomes on the left-side of the image, after being hit by Doppler ultrasound, can be seen.

 

As the amplitude of the ultrasound pulses is stepped up gradually, the ringing effect was found to increaseindicating that stable cavitation sets in at low amplitude and continues to occur even at higher amplitudes. On the other hand, the ultrasound pulses begin to pop the vesicles substantially only at higher amplitudes, indicating that there is a sweet spot where stable cavitation occurs without inertial cavitation setting in. Concurrent with the popping of the vesicles, there is also a higher disappearance of contrast on the ultrasound images.

 

In further studies, we will examine how the stable cavitation and inertial cavitation activities correlate with the amount of drug release from echogenic liposomes and the delivery of these drug molecules into blood vessels. We will also examine the vessels for cellular damage and optimize the range of amplitudes over which the drug release and delivery is enhanced without negative biological effects.