Sonoporation with phase-shift nanoemulsions: an in vitro study
Burgess, Mark Thomas
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Acoustic cavitation (i.e. acoustically stimulated microbubble activity) has gained interest in the biomedical community due to its ability to locally concentrate mechanical forces inside the body. Biological structures in close proximity experience stresses that temporally disrupt their normal function and allow passage of material that would otherwise be impermeable. Examples include blood-brain barrier disruption, enhanced penetration of drugs into tumors, disruption of the blood vessel endothelium, and permeabilization of cell membranes (i.e. sonoporation). The goal of this thesis was to investigate a new class of acoustic cavitation nuclei for sonoporation called phase-shift nanoemulsions (PSNE). Ultrasound can be used to nucleate, or phase-shift PSNE into microbubbles with a process termed acoustic droplet vaporization (ADV). Specifically, the focus was to use PSNE for delivery of small interfering RNA (siRNA) to an in vitro cell suspension using sonoporation. Small interfering RNA is an exogenous RNA molecule and has gained increased attention due to its ability to knockdown specific proteins central to disease progression. Results showed that siRNA delivery with PSNE is possible with high uptake efficiency (i.e. ratio of the number of cells with uptake to the number of cells originally). Uptake was highly dependent on the amount of acoustic cavitation activity generated from PSNE. The acoustic emissions from individual PSNE were explored to understand the microbubble dynamics following ADV. Results showed that PSNE immediately undergo an explosive growth and collapse at the ADV threshold, and the maximum size of the microbubble depends on the ultrasound frequency. This led to the hypothesis that the sonoporation efficiency with PSNE is governed by the choice of frequency. Lower frequencies were shown to expand microbubbles to larger maximum radii, which in turn caused more energetic collapses leading to cell death. This explains the lower uptake efficiencies at lower frequencies (39.45% at 1 MHz and 46.62% at 2.5 MHz), compared to the relatively high uptake efficiency at 5 MHz (66.81%). In general, uptake efficiencies > 50% have rarely been achieved with current sonoporation methods and these results are a significant improvement. PSNE could also serve as a unique platform for numerous other therapeutic ultrasound applications that utilize the mechanical effects of acoustic cavitation. The frequency-dependent control over the microbubble dynamics following ADV could provide a way to tune the level of stress experienced by biological structures.