Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the bloodÀbrain and bloodÀspinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments.

The lytic recombinant tissue plasminogen activator (rt-PA) is the only drug approved by the Food and Drug Administration for treating ischemic stroke. Less than 40% of patients with large vessel occlusions who are treated with rt-PA have improved blood flow. However, up to 6% of all patients receiving rt-PA develop intracerebral hemorrhage. Predicting the efficacy of rt-PA treatment a priori could help guide therapeutic decision-making, such that rt-PA is administered only to those individuals who would benefit from this treatment. Clot composition and structure affect the lytic efficacy of rt-PA and have an impact on elasticity. However, the relationship between clot elasticity and rt-PA lytic susceptibility has not been adequately investigated. The goal of this study was to quantify the relationship between clot elasticity and rt-PA susceptibility in vitro. Human and porcine highly retracted and mildly retracted clots were fabricated in glass pipettes. The rt-PA lytic susceptibility was evaluated in vitro using the percent clot mass loss. The Young’s moduli of the clots were estimated using ultrasound-based single-track-location shear wave elasticity imaging. The percent mass loss in mildly retracted porcine and human clots (28.9 ± 6.1% and 45.2 ± 7.1%, respectively) were significantly higher (p < 0.05) than those in highly retracted porcine and human clots (10.9 ± 2.1% and 25.5 ± 10.0%, respectively). Furthermore, the Young’s moduli of highly retracted porcine and human clots (5.33 ± 0.92 kPa and 3.21 ± 1.97 kPa, respectively) were significantly higher (p < 0.05) than those of mildly retracted porcine and human clots (2.66 ± 0.55 kPa and 0.79 ± 0.21 kPa, respectively). The results revealed an inverse relationship between the percent clot mass loss and Young’s modulus. These findings motivate continued investigation of ultrasound-based methods to assess clot stiffness in order to predict rt-PA thrombolytic efficacy.

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy ( p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone ( p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

Adjuvant ultrasound at 2 MHz with or without an ultrasound contrast agent improves the rate of thrombus resolution by recombinant tissue plasminogen activator (rt-PA) in laboratory and clinical studies. A sub-megahertz approach can further expand this therapy to a subset of patients with an insufficient temporal bone window, improving efficacy in unselected patient populations. The aim of this study was to determine if a clinical ultrasound contrast agent (UCA), Definity, and 220 kHz pulsed ultrasound accelerated rt-PA thrombolysis in a preclinical animal model of vascular occlusion. The effect of Definity and ultrasound on thrombus clearance was first investigated in vitro and subsequently tested in a xenographic porcine cerebral thromboembolism model in vivo. Two different microcatheter designs (end-hole, multi-side-hole) were used to infuse rt-PA and Definity at the proximal edge or directly into clots, respectively. Sonothrombolysis with Definity increased clot mass loss relative to saline or rt-PA alone in vitro, only when rt-PA was administered directly into clots via a multi-side-hole microcatheter. Combined treatment with rt-PA, Definity, and ultrasound in vivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline. In this porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity increased the probability of early successful reperfusion with rt-PA.

Ultrasound-enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis is under development as an adjuvant to ischemic stroke therapy. The goal of this study was to design a pulsed ultrasound (US) exposure scheme that reduced intracranial constructive interference and tissue heating, and maintained thrombolytic efficacy relative to continuous wave (CW) insonation. Three 220 kHz US schemes were evaluated, two pulsed insonation schemes (15 cycles, 68 µs pulse duration, 33% or 62.5% duty cycle) and an intermittent CW insonation scheme (50 s active, 30 s quiescent) over a 30-min treatment period. An in silico study using a finite-difference model of transcranial US propagation was performed to estimate the intracranial acoustic field and temperature rise in the skull for each insonation scheme. In vitro measurements with flow were performed to assess thrombolysis using time-lapse microscopy. Intracranial constructive interference was not reduced with pulsed US using a pulse length of 15 cycles compared to intermittent CW US. The 33.3% duty cycle pulsed US scheme reduced heating in the temporal bone as much as 60% relative to the intermittent CW scheme. All insonation schemes promoted sustained stable cavitation in vitro and augmented thrombolysis compared to rt-PA alone (p < 0.05). Ultraharmonic (UH) and harmonic cumulative energy over a 30 min treatment period was significantly higher (p < 0.05) for the intermittent CW US scheme compared to either pulsed US scheme. Despite the difference in cavitation emissions, no difference was observed in the clot lysis between the three US schemes. These findings demonstrate that a 33.3% duty cycle pulsed US scheme with a 15-cycle burst can reduce bone heating and achieve equivalent thrombolytic efficacy as an intermittent CW scheme.

Predicting thrombolytic susceptibility to recombinant tissue plasminogen activator (rt-PA) a priori could help guide clinical decision-making during acute ischemic stroke treatment and avoid adverse off-target lytic effects. The composition and structure of clots impact their mechanical properties and rt-PA thrombolytic efficacy. The goal of this study was to determine the relationship between clot elasticity and rt-PA thrombolytic efficacy in vitro. Human and porcine retracted and unretracted clots were fabricated in glass pipettes. Clots were embedded in agar phantoms, and their Young’s moduli were estimated using single-track-location shear wave elasticity imaging. The rt-PA thrombolytic efficacy was evaluated in vitro using the percent clot mass loss. The Young’s moduli of unretracted porcine and human clots (1.68±0.18 kPa and 0.72±0.13 kPa, respectively) were significantly lower (p&lt;0.05) than those of retracted porcine and human clots (4.96±1.07 kPa and 3.38±1.82 kPa, respectively). The percent mass loss of unretracted porcine and human clots (28.9±6.1% and 45.2±7.1%, respectively) were significantly higher (p&lt;0.05) than those of retracted porcine and human clots (10.9±2.1% and 25.5±10.0%, respectively). The results revealed a linear inverse correlation between the Young’s moduli and percent clot mass loss (R2 = 0.95, p = 0.025), suggesting that clot stiffness may serve as a surrogate metric for rt-PA thrombolytic susceptibility.

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