A Comparison of Sonothrombolysis in Aged Clots between Low-Boiling-Point Phase-Change Nanodroplets and Microbubbles of the Same Composition

Abstract

We present enhanced cavitation erosion of blood clots exposed to low-boiling-point (-2°C) perfluorocarbon phase-change nanodroplets and pulsed ultrasound, as well as microbubbles with the same formulation under the same conditions. Given prior success with microbubbles as a sonothrombolysis agent, we considered that perfluorocarbon phase-change nanodroplets could enhance clot disruption further beyond that achieved with microbubbles. It has been hypothesized that owing to their small size and ability to penetrate into a clot, nanodroplets could enhance cavitation inside a blood clot and increase sonothrombolysis efficacy. The thrombolytic effects of lipid-shell-decafluorobutane nanodroplets were evaluated and compared with those of microbubbles with the same formulation, in an aged bovine blood clot flow model. Seven different pulsing schemes, with an acoustic intensity (I_SPTA) range of 0.021-34.8 W/cm² were applied in three different therapy scenarios: ultrasound only, ultrasound with microbubbles and ultrasound with nanodroplets (n = 5). Data indicated that pulsing schemes with 0.35 W/cm² and 5.22 W/cm² produced a significant difference (p < 0.05) in nanodroplet sonothrombolysis performance compared with compositionally identical microbubbles. With these excitation conditions, nanodroplet-mediated treatment achieved a 140% average thrombolysis rate over the microbubble-mediated case. We observed distinctive internal erosion in the middle of bovine clot samples from nanodroplet-mediated ultrasound, whereas the microbubble-mediated case generated surface erosion. This erosion pattern was supported by ultrasound imaging during sonothrombolysis, which revealed that nanodroplets generated cavitation clouds throughout a clot, whereas microbubble cavitation formed larger cavitation clouds only outside a clot sample.

Keywords: Cavitation; Clot; Microbubbles; Nanodroplets; Sonothrombolysis.

Fig. 1.

Experimental setup. (a) Front view of a flow model. (b) A blood clot sample was fixed by a nylon mesh; the yellow arrow denotes flow direction (scale bar = 10 mm). (c) Side view of the setup. (d) A aged clot sample (288 mg).

Fig. 2.

Schematic of cavitation imaging setup. The cavitation clouds from nanodroplet-mediated pulsed ultrasound (group 3) were monitored and compared with the microbubble cavitation clouds (n = 9).

Fig. 3.

In vitro thrombolytic efficacy test results in terms of percent clot-mass reduction during 10 min treatment (n = 5) with insonation groups 1–4. The asterisk (*) indicates a significant difference (p < 0.05).

Fig. 4.

Passive cavitation detection results (n = 30). (a) Stable cavitation level and (b) inertial cavitation level. The asterisk (*) indicates a significant difference (p < 0.05).

Fig. 5.

Representative captured images of clot erosion by US+ND treatment (group 3). A yellow arrow indicates flow direction (scale bar = 5 mm). Within 10 s of the treatment, micro-sized erosion was observed (light-blue solid arrow).

Fig. 6.

Cavitation imaging results. (a) The US+ND cavitation zone (yellow-green cloud) is located inside the clot. (b) US+MB shows cavitation clouds outside of the clot. The red arrows denote the propagation direction of therapeutic pulses, red-dotted circles indicate approximate inner tube diameter and light-blue arrows indicate a nylon mesh. (c) A comparison of the in-clot cavitation metric (n = 9).

Fig. 7.

In vitro thrombolytic efficacy test results with insonation groups 5, 6 and 7. (a) Percent clot-mass reduction during 10 min treatment (n = 5), (b) passive cavitation (stable) detection results and (c) inertial cavitation level (n = 30). The asterisk (*) indicates a significant difference (p < 0.05).

Fig. 8.

Representative captured images of clot erosion by US+ND and US+MB treatment (group 7). A yellow arrow indicates flow direction (scale bar = 5 mm). After 20 min of treatment, US+ND shows clear multiple erosions, whereas micro-size erosions are observed from US + MB treatment (light-blue solid arrow).