Temporal Stability of Lipid-Shelled Microbubbles During Acoustically-Mediated Blood-Brain Barrier Opening

Abstract

Non-invasive blood-brain barrier (BBB) opening using focused ultrasound (FUS) is being tested as a means to locally deliver drugs into the brain. Such FUS therapies require injection of pre-formed microbubbles, currently used as contrast agents in ultrasound imaging. Although their behavior during exposure to imaging sequences has been well-described, our understanding of microbubble stability within a therapeutic field is still not complete. Here, we study the temporal stability of lipid-shelled microbubbles during therapeutic FUS exposure in two timescales: the short timescale (i.e., µs of low-frequency ultrasound exposure) and the long timescale (i.e., days post-activation). We first simulated the microbubble response to low-frequency sonication, and found a strong correlation between viscosity and fragmentation pressure. Activated microbubbles had a concentration decay constant of 0.02 d −1 but maintained a quasi-stable size distribution for up to 3 weeks (<10% variation). Microbubbles flowing through a 4-mm vessel within a tissue-mimicking phantom (5% gelatin) were exposed to therapeutic pulses (f c : 0.5 MHz, peak-negative pressure: 300 kPa, pulse length: 1 ms, pulse repetition frequency: 1 Hz, n = 10). We recorded and analyzed their acoustic emissions, focusing on emitted energy and its temporal evolution, alongside the frequency content. Measurements were repeated with concentration-matched samples (10 7 microbubbles/ml) on day 0, 7, 14, and 21 after activation. Temporal stability decreased while inertial cavitation response increased with storage time both in vitro and in vivo, possibly due to changes in the shell lipid content. Using the same parameters and timepoints, we performed BBB opening in mice (n = 3). BBB opening volume measured through T1-weighted contrast-enhanced MRI was equal to 19.1 ± 7.1 mm 3 , 21.8 ± 14 mm 3 , 29.3 ± 2.5 mm 3 , and 38 ± 20.1 mm 3 on day 0, 7, 14, and 21, respectively, showing no significant difference over time (p-value: 0.49). Contrast enhancement was 24.9 ± 1.7%, 23.7 ± 11.7%, 28.9 ± 5.3%, and 35 ± 13.4%, respectively (p-value: 0.63). In conclusion, the in-house Pouliopoulos et al.Microbubble Stability in Ultrasound Therapy made microbubbles studied here maintain their capacity to produce similar therapeutic effects over a period of 3 weeks after activation, as long as the natural concentration decay is accounted for. Future work should focus on stability of commercially available microbubbles and tailoring microbubble shell properties toward therapeutic applications.

Recent Advances on Ultrasound Contrast Agents for Blood-Brain Barrier Opening with Focused Ultrasound

“…Lipid-shelled microbubbles are highly responsive to ultrasound, while the polymer and protein-shelled microbubbles are very stable and require higher acoustic pressure to undergo cavitation [ 24 , 139 ]. PEG-surfactants are systematically used with phospholipid shells due to their ability to appreciably lower surface tension [ 102 ]. Other surfactant shells have also been explored in echogenic contrast agent fabrication [ 128 ].…”

“…Pouliopoulos et al compared different lipid shell microbubbles shelled on their acoustic stability in vitro [ 102 ]. The shells were composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N -[methoxy(polyethylene glycol)2000] (DSPE-PEG2000) with different molar ratio (6:1; 9:1; and 12:1).…”

“…The energy transmitted by the excited bubble was processed to appraise the different shell acoustic stabilities. Eventually, a 9:1 ratio was found to provide higher acoustic stability [ 102 ]. DSPC:DSPE-PEG2000 at a molar ratio of 9:1 is the most commonly formulated lipid shell for microbubble self-made for BBB disruption.…”

Mapping Knowledge Structure and Research Frontiers of Ultrasound-Induced Blood-Brain Barrier Opening: A Scientometric Study

“…Therefore, the researchers from the USA were the main driving force, as evidenced by four of the top 5 most productive authors being from the USA. Hynynen K from Brigham and Women's Hospital, Liu H L from Chang Gung Memorial Hospital/Chang Gung University (Liu et al, 2010a , b ), and Konofagou E E (Konofagou, 2012 ; Pouliopoulos et al, 2020 ) from Columbia University contributed the most publications. However, in terms of authors' cooperation analysis, the centrality index for each author was <0.1, and quite a small number of connection links were observed in this network map, which reflected that there was little collaboration between different research teams.…”

“…As we all know, microbubble preparations were originally developed and exploited as ultrasound contrast agents, which were used for the enhancement of ultrasound imaging and detection, due to their ability to generate an enhanced echogenic effect. Subsequently, microbubbles were observed to be capable of generating cavitation effect in ultrasonic fields, which could be further classified into stable and inertial cavitation: the former generated a microstreaming at the relatively lower ultrasound intensities, and the latter produced a shock wave and microjet in response to high-pressure fields (Hynynen et al, 2001 , 2005 ; Pouliopoulos et al, 2020 ). Based on this characteristic, it was quickly realized that ultrasound combined with microbubble preparations may offer a suitable means of delivering small molecule drugs, genes, or other macromolecules into the brain by temporarily opening the tight junction of BBB (Unger et al, 2004 ; Sheikov et al, 2008 ).…”