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Most ultrasound-based therapies require delivery via sonoporation, the process by which otherwise impermeable membranes are made permeable in the presence of ultrasound and microbubbles. Despite its widespread use, the physical mechanism(s) of sonoporation is not fully understood. Others have shown that low frequency ultrasound (LFUS, 20-100 kHz) can facilitate membrane permeability in the absence of microbubbles. Microbubble response to ultrasound has been proven sensitive to shell-chemistry, pointing to the probability that membrane physical properties play a role in membrane response to ultrasound. This work examines the hypothesis that ultrasound-induced transport correlates with membrane phase behavior.We present a quantitative study of LFUS-induced release from large unilamellar vesicles (LUVs). Three well-documented systems are studied: binary, raft-forming ternary, and non-raft-forming ternary. Both extents and rates of release are measured using steady state fluorescence microscopy and relief-of-self-quenching of calcein assays. Dynamic light scattering (DLS) is used to determine vesicle destruction. Release kinetics are fit with simple mathematical models that account for diffusion and bilayer destruction.Response of the binary system to ultrasound proved insensitive to lipid-phase behavior. In the raft-forming ternary systems, as membrane phase changes toward liquid-ordered, the membrane becomes increasingly resistant to release. Two mechanisms of release are evident in ld samples, diffusion and destruction. lo samples do not exhibit vesicle destruction and fit well to the diffusion-only model.Release in the non-raft-forming ternary system decreases as cholesterol increases within purely liquid regions. The most rapid release occurred in samples with some so phase, indicating LFUS sensitivity to the presence of so phase. Both diffusion-only and destruction-only models fit well to release profiles, and DLS data did not indicate clear evidence of destruction.We interpret these results to mean ultrasound-induced permeation correlates directly with phase behavior in complex systems and is not sensitive to liquid-liquid coexistence. However, release is sensitive to solid-liquid coexistence. In raft-forming systems membrane phase has a stronger influence then cholesterol mole fraction. In non-raft-forming systems release correlates with cholesterol, except in the presence of so phase. Models reveal multiple mechanisms can describe release independently, though in some cases a combination of mechanisms best describes release. The findings herein might prove useful in designing and developing therapies based on ultrasound and membrane interactions. |
