The advancement of ultrasound-mediated therapy has stimulated the development of drug-loaded microbubble agents that can be targeted to a region of interest through an applied magnetic field prior to ultrasound activation. However, the need to incorporate therapeutic molecules while optimizing the responsiveness to both magnetic and acoustic fields and maintaining adequate stability poses a considerable challenge for microbubble synthesis. The aim of this study was to evaluate three different methods for incorporating iron oxide nanoparticles (IONPs) into phospholipid-coated microbubbles using (1) hydrophobic IONPs within an oil layer below the microbubble shell, (2) phospholipid-stabilized IONPs within the shell, or (3) hydrophilic IONPs noncovalently bound to the surface of the microbubble. All microbubbles exhibited similar acoustic response at both 1 and 7 MHz. The half-life of the microbubbles was more than doubled by the addition of IONPs by using both surface and phospholipid-mediated loading methods, provided the lipid used to coat the IONPs was the same as that constituting the microbubble shell. The highest loading of IONPs per microbubble was also achieved with the surface loading method, and these microbubbles were the most responsive to an applied magnetic field, showing a 3-fold increase in the number of retained microbubbles compared to other groups. For the purpose of drug delivery, surface loading of IONPs could restrict the attachment of hydrophilic drugs to the microbubble shell, but hydrophobic drugs could still be incorporated. In contrast, although the incorporation of phospholipid IONPs produced more weakly magnetic microbubbles, it would not interfere with hydrophilic drug loading on the surface of the microbubble.

Tethers are thin tubes of lipids (~20-200 nm in diameter) that form when membranes are subjected to a point force. Tether dynamics are important to a myriad of biological processes including white blood cell adhesion and transport of intracellular material between neighboring cells. To understand the dynamics of tether formation more fully, we investigated the dependence of the force needed to create a tether on the rate of force change (loading rate). To conduct these experiments, a microfabricated magnetic force transducer was used to generate well-controlled and localized magnetic force profiles. Tethers were formed off the surface of microaspirated giant unilamellar vesicles (GUVs) attached to magnetic beads. We discovered a strong correlation between the threshold force of tether formation and the applied force ramp, with the force changing from <10 pN at low loading rates to~50 pN at high loading rates. At slow loading rates, the threshold force changes weakly with ln (loading rate), while at high loading rates a steeper dependence is observed. The experimental data can be fit to a energetic model based on Kramer's theory, similar to models used to describe membrane rupture. The model predits that tether formation involves passage over two energy barriers and enbales characterization of the characteristic forces and timescales associated with these barriers. This new tool for dynamic studies of membrane mechanics may further be extended to study how tethers form off of flowing cells or how phase regimes, induced by the presence of cholesterol, influence membrane dynamics.

Blood-brain barrier (BBB) disruption mediated by ultrasound and microbubbles (US-BBBD) is a promising strategy for non-invasive and targeted delivery of therapeutics to the brain. In US-BBBD, treatment control is achieved by externally monitoring acoustic emissions (AE) and adjusting ultrasound parameters in real-time to avoid AE associated with damage. Recent work suggests that AE may also provide insight regarding the extent of BBB opening and BBB recovery time. The mechanisms underlying BBB opening and recovery, however, are largely not understood. To investigate US-BBBD mechanisms with regard to AE, we developed an in vitro platform for monitoring both BBB integrity and AE during US-BBBD. Temporally resolved BBB integrity monitoring was achieved using a microfluidic BBB-on-a-chip device with integrated trans-endothelial electrical resistance (TEER) measurements. Well-characterized ultrasound exposure and AE monitoring were achieved using a focally aligned high-intensity focused ultrasound transducer and passive cavitation detector. In addition to recording TEER and AE data, our platform is compatible with fluorescence microscopy during ultrasound exposure, providing further insight into US-BBBD mechanisms. This work further demonstrates potential for in vitro screening of cavitation agents and/or therapeutics for novel US-BBBD applications and strategies.

Here we investigate the cavitation phenomenon at a lipid interface of multilaminar vesicles (MLVs) subjected to acoustic shock waves. The lipid membranes contain a fluorescent dye, Laurdan, which produces a fluorescence emission sensitive to the thermodynamic state of the interface. Fluorescence emissions were measured at 438nm and 470nm using two photomultiplier tubes (with 8 MHz bandwidth) from which the temporal evolution of the interface’s thermodynamic state was determined with submicrosecond resolution. Acoustic emissions were recorded simultaneously in order to detect the presence of cavitation. Different lipids were used to prepare the MLVs in order to observe cavitation phenomenon as a function of the state of the interface. It was deduced that the interface behaves as an adiabatic system decoupled from the bulk, where the entropy increase due to vaporization during cavitation is compensated by the entropy decrease resulting from condensation and dehydration of the lipids. These results show that cavitation physics critically depends on the thermodynamics of the interface. While applied here on a simple system of pure lipid MLVs, the thermodynamic approach is applicable to native biological membranes and cavitation phenomenon in general. [Work supported by UK EPSRC EP/L024012/1.]

High-intensity focused ultrasound (HIFU) is often used to create lesions, or regions of tissue destruction due to heating and cavitation activity, most often in tumors or other diseased tissues. However, the acoustic properties of tissues denatured by heat are not very different from those of untreated tissue, making lesion detection and quantification difficult by ultrasound alone. Autofluorescence refers to the broadband emission of light within materials that are stimulated by narrowband incident light, typically from a laser. It is a common characteristic of lipids, proteins, and other biomolecules, and the “autofluorescence spectrum” is a function of the state of the material. We have examined irreversible shifts in the dominant autofluorescence spectra of proteins denatured by HIFU heating. These shifts appear to be related to protein conformational changes due to denaturation. We report on the feasibility of using optical autofluorescence as a means of quantifying in vitro lesion formation by HIFU for optically accessible tissues.