How to create tiny vapour bubbles near lipid membranes using sound waves

A scientific and impact summary of the paper titled “Thermodynamic state of the interface during acoustic cavitation in lipid suspension” Phys. Rev. Materials 3, 055602 — Published 9 May 2019

The main significance of this paper

This paper shows that it is much easier to rupture (cavitate) water if it contains lipids that are about to “melt”; water with lipids in a frozen or liquid state is harder to cavitate. Second; the paper shows that when a cavitation bubble expands the lipids at the surface of the bubble can condense and “freeze”.

Medical Applications of Acoustic Cavitation

Acoustic cavitation has a number of medical application. In diagnostic ultrasound imaging, when visualisation of blood flow is important then small microbubbles of gas are injected into the bloodstream (and yes it is safe to inject small microbubbles of gas) and the response of these bubbles makes the vessels light up. This is widely accepted in the imaging community. An emerging use of the microbubbles is to load them with drugs and then if they are hit with a specific ultrasound pulse they will pop and release the drug to a localised position. This is a technology undergoing trials at present.

Ultrasound can also be used therapeutically to destroy soft tissue (both cancerous and benign) using high intensity focussed ultrasound and breaking kidney stones using shock waves (lithotripsy). Cavitation plays an important role in most of these therapeutic applications as it converts the relatively benign ultrasound energy into a highly localised and intense event. Lithotripsy is a mature and well-accepted technology; high intensity focused ultrasound is approved for treating uterine fibroids and prostate and is emerging into numerous medical applications.

Now, researchers are also trying to use cavitation for subtle control of cellular functions that is based on reversible biophysical interactions. So for example, controlling the permeability of cell membranes and cell junctions or controlling the electrical polarization of the cell membranes, which will have applications in enhanced drug delivery or altering other biological functions of the membrane. Clinical trials have shown that such applications are possible. However, the mechanisms are poorly understood, which is hampering clinical translation of such approaches. This study contributes to this gap.

A previous blog covered the various applications and prospects of medical ultrasound in more detail. https://medium.com/@Shamits/a-sound-future-for-noninvasive-therapies-ac487ea03977

The therapeutic benefits of using acoustic cavitation.

As mentioned above, cavitation bubbles can convert the ultrasound energy into highly localised mechanical and thermal energy which can be used to ablate or fractionate tissue. This is a key advantage of acoustic cavitation in the therapeutic applications mentioned above.

The use of bubbles to help drugs penetrate cells is more complex and this work contributes particularly to that application. The growth and collapse of cavitation results on mechanical forces on nearby cells; and the cell walls (which are made of lipids-similar to those studied here) respond to these resulting in changes in permeability such that drugs can then pass into the cells. Further, for the cells that line the vessel in the brain (the so-called blood-brain barrier) the action of microbubbles to ultrasound results in a temporary opening of the barrier so that drugs can be delivered to brain tissue that is otherwise inaccessible. This study confirms that cavitation can indeed result in significant state changes in lipid membranes and gives a physical basis for ultrasound-microbubble mediated drug delivery.

The difference from previous cavitation models

Previous bubble models have incorporated the effects of surfactants and lipids based only on their surface or interfacial tension. However, the complete first law of energy conservation for the interface has an entropy component as well. I.e. the interface can store heat. Current models neglect the entropy or the heat content of the interface. Without accounting for the interfacial entropy the previous bubble models are incapable of explaining the two experimental observations made in this study: (i) minimum in cavitation threshold at the phase transitions of lipids, and (ii) condensation of lipids upon expansion of the cavitation bubbles. Details below in the question regarding phase transitions.

Fluorescence changes for real-time observation of the membrane during cavitation

The fluorescent marker reports the local dielectric constant of the material, which is directly related to the conformational state of the lipid membrane. The more disordered the membrane is, the more redshifted is the emission spectrum of the marker. Thus we can observe in real-time (sub-microsecond resolution), how the order of the lipid membrane changes during cavitation. This observation allowed us to infer during cavitation when the membrane is condensing or fluidizing and by how much.

The surprising role of lipid phase transitions

Cavitation is a stochastic process in which the fluid is ruptured (vaporised) by a negative pressure (also known as tensile stress). The cavitation will initiate at points of weakness in the fluid which are dependent on local fluctuations at a molecular level. At a phase transition, the fluctuations are much stronger (think of the surface of boiling water) and so if a lipid is close to phase transition it will contribute to many more potential weak spots for cavitation to initiate. Once a nucleation event has happened the negative pressure results in rapid expansion into a cavitation bubble.

The second effect is during the expansion phase where both water and lipids will vapourise to fill in the growing cavity. In order to vaporise they require heat energy (the latent heat). For the time scales investigated here all the latent heat has to come from the interface and so almost non-intuitively the lipids left behind freeze in order to give energy to the molecules to vapourise.

Applications

I think the most immediate application is in the design of lipid nanoparticles, which can be easily made to be near a phase transition under physiological conditions. We have applied this approach recently to design lipid nanoparticles for intracellular delivery of mRNA(2) using ultrasound. We have now shown that such nanoparticles will be most readily affected by cavitation. The study also shows that, at least in principle, by tuning ultrasound parameter one can control the rate of expansion of cavitation bubble, allowing us to either fluidize the lipid membrane (at slow expansion rate) or condense it (fast expansion rate). However, we could not resolve the inversion point where the behaviour changes due to the limitations of our setup.

Finally, phase transitions occur in native biological membranes too, however currently it’s not trivial to measure the state of the membranes in vivo and further advances in current technology are needed that will allow us to access the state of the membrane and thus allowing us to better tune the ultrasonic parameters for more specific effects.

Future work

The study underlines the importance of the thermodynamic state of the interface in determining the biophysical effects of cavitation. We have started applying this knowledge in designing biomaterials, where we can control the material properties to control the state. Thus, for example, designing lipid nanoparticles that are closed to phase transition which makes them most likely to cavitate. This also has implications for more advanced applications where cavitation interacts with the native membrane. If the state of the biological membrane can be measured in-vivo, then acoustic parameters can be designed to interact strongly and specifically with the target. Thus measuring the state of the membranes provides an important goal the future of diagnostics that can work in concert with the therapeutic ultrasound, helping optimise the biophysical effects of ultrasound.

References

1. Shrivastava S, Cleveland RO, Schneider MF (2018) On measuring the acoustic state changes in lipid membranes using fluorescent probes. Soft Matter 14:9702–9712.

2. Zhang J, Shrivastava S, Cleveland RO, Rabbitts TH (2019) Lipid-mRNA Nanoparticle Designed to Enhance Intracellular Delivery Mediated by Shock Waves. ACS Appl Mater Interfaces:acsami.8b21398.