Shaken and Stirred: Mechanisms of Ultrasound-Enhanced Thrombolysis

Abstract

The use of ultrasound and microbubbles as an effective adjuvant to thrombolytics has been demonstrated in vitro, ex vivo, and in vivo. However, the specific mechanisms of ultrasound-enhanced thrombolysis (UET) have yet to be elucidated. We present visual observations illustrating two mechanisms of UET: acoustic cavitation and radiation force. An in vitro flow model was developed to observe human whole blood clots exposed to human fresh-frozen plasma, rt-PA (0, 0.32, 1.58, or 3.15 μg/mL), and the ultrasound contrast agent Definity® (2 μL/mL). Intermittent, continuous-wave, ultrasound (120 kHz, 0.44 MPa peak-to-peak pressure) was used to insonify the perfusate. Ultraharmonic (UH) emissions indicative of stable cavitation were monitored with a passive cavitation detector. The clot was observed with an inverted microscope, and images were recorded with a charge-coupled device (CCD) camera. The images were post processed to determine the time-dependent clot diameter and root-mean-square velocity of the clot position. Clot lysis occurred preferentially surrounding large, resonant-sized bubbles undergoing stable oscillations. UH emissions from stable cavitation were found to correlate with the lytic rate. Clots were observed to translate synchronously with the initiation and cessation of the ultrasound exposure. The root-mean-square velocity of the clot correlated with the lytic rate. These data provide visual documentation of stable cavitation activity and radiation force during sub-megahertz sonothrombolysis. The observations of this study suggest that the process of clot lysis is complex, and both stable cavitation and radiation force are mechanistically responsible for this beneficial bioeffect in this in vitro model.

Microfluidic manufacture of rt-PA -loaded echogenic liposomes

“…A wide range of particle concentrations has been reported in literature for studies on sonothrombolysis. Our group has demonstrated thrombolysis in vitro for approximately 1 × 10 6 microbubbles/ml (Bader et al 2015a, b), two orders of magnitude lower than the liposome concentration achieved in this work. Shih et al have reported microbubble generation rates as high as 3 × 10 6 per s (Shih et al 2013).…”

“…However, microbubbles larger 7 µm are filtered by the lungs and thus for cavitation nucleation for enhancement of rt-PA thrombolysis. Resonant bubbles of 20–50 µm could be formed by coalescence of smaller bubbles liberated from µtELIP experiencing Bjerkness forces (Bader et al 2015a, b). Therefore, µtELIP could be tested for sonothrombolysis at sub-megahertz frequencies.…”

“…Perfluorocarbon gases are known to improve the lifetime of echo contrast agents due to low solubility in aqueous media and low diffusivity through lipid shells, and are used in commercial contrast agents such as Definity ® (Raymond et al 2014). Studies have shown that the cavitation activity from rt-PA loaded ELIP with an air core is lower than Definity ® , suggesting that incorporating perfluorocarbon gas within ELIP instead of air may enhance their cavitation activity and thrombolytic efficacy (Bader et al 2015a, b). The shell friction parameter (S F ) for µtELIP is significantly higher than Definity ® or batch-produced rt-PA-loaded ELIP (Table 1).…”

Sonothrombolysis

“…Frenkel et al (2006), using both an in-vitro clot model and a numerical model to calculate clot displacement, found a correlation between thrombolytic efficacy and acoustic radiation force at 1 MHz. Bader et al (2015) found a correlation between the root-mean-square velocity of displaced in-vitro human whole blood clots and the lytic rate with sub-megahertz insonation. Radiation force is known to act on bubbles (Leighton 1994), and several groups have shown microbubbles forced into clots in-vitro (Caskey et al 2009; Acconcia et al 2013; Everbach and Guarini 2013) (Fig.…”

Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis

“…Conversely, control MBs or ACPP-MBs, in the presence of thrombin-inhibiting hirudin, should have a lower binding to the clot, limited to the nonspecific adhesion of MBs. 50 Since the clot and transducer are mechanically fixed, images are perfectly coregistered throughout the experiment. We subtracted the baseline US images acquired before MB infusion from the images acquired after the final saline wash, and the signal on a pixel-by-pixel basis was converted to a color-coded heat map and overlaid on the baseline grayscale B-mode images (Figure 6A).…”