Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

Deep vein thromboses (DVT) are prevalent and challenging to treat, particularly because DVT clots are often older, denser, stiffer, and retracted, making them less responsive to standard therapies. Systemic tPA can require long infusion times and high doses, increasing hemorrhagic risk; mechanical thrombectomy can damage endothelium or generate embolic debris. Sonothrombolysis—ultrasound-enhanced thrombolysis—leverages cavitation (stable and inertial) to mechanically disrupt clots and enhance drug penetration, and has shown efficacy in unretracted clots with microbubbles. However, MB-mediated sonothrombolysis is less effective in retracted clots, and external high-intensity ultrasound approaches (HIFU, histotripsy) require high peak negative pressures and large external transducers that limit applicability. Nanodroplets (100–300 nm) can penetrate dense, low-porosity retracted clots and, upon activation, form microchannels that enhance mechanical disruption and tPA diffusion. Low-boiling point phase change NDs have vaporization thresholds near ~1 MPa, potentially enabling activation within safety limits. The authors previously developed a sub-megahertz forward-viewing intravascular (FVI) transducer enabling site-specific insonation. This study investigates whether the FVI transducer can activate low-boiling point nanodroplets and, combined with tPA, improve sonothrombolysis in retracted clots in vitro.

Prior work shows sonothrombolysis with MBs enhances thrombolysis in unretracted clots via cavitation-induced microstreaming and improved drug diffusion. In retracted clots, MBs have limited efficacy due to low porosity. HIFU and histotripsy have lysed retracted clots in preclinical studies but at high PNPs (up to ~15 MPa) and with large external transducers, limiting use in certain vascular territories (e.g., behind ribs/lungs). Nanodroplets have been used for imaging, drug delivery, and thrombolysis; conventional ND activation often needs 3–5 MPa. Low-boiling point NDs can reduce activation thresholds (~1 MPa), potentially improving safety and feasibility. Prior ND+HIFU thrombolysis studies in fresh, unretracted clots achieved 25–80% mass loss at PNPs of 2–8 MPa. There remains a gap in intravascular ultrasound approaches using NDs, particularly for retracted clots at lower, clinically acceptable PNPs.

FVI transducer: A forward-viewing intravascular transducer (similar to prior work) was fabricated from seven stacked layers of PZT-5A with E-solder 3022 bonding and an alumina epoxy matching layer. Aperture: 1.5 mm. Center frequency: 700 kHz; bandwidth: 445 kHz–1.05 MHz; impedance: 225 Ω; capacitance: 1.43 nF. Peak-to-peak driving voltages of 15, 35, 50, and 70 V yielded PNPs of 0.3, 0.6, 0.9, and 1.2 MPa, respectively (sensitivity 0.02 MPa/V; driving power 11 W). The transducer was integrated into a catheter with an injection lumen (overall catheter diameter 3 mm).

Passive cavitation detection (PCD) for ND activation: To determine the minimum PNP for activation of low-boiling point phase change NDs, the FVI transducer was driven at 700 kHz with 10 ms pulse length and 20 cycles. Solutions of NDs (10^8 ND/ml) or phosphate-buffered saline (PBS) were injected through the integrated catheter at 0.1 ml/min using a syringe pump. PNP was varied from 0.3 to 1.2 MPa. Cavitation emissions were recorded with a hydrophone (HGL-0085, ONDA). Three RF acquisitions per condition were obtained. Stable cavitation dose was computed as the area under the frequency spectrum at the second harmonic (2f0 ± 0.5f0). Inertial cavitation dose was computed as the area under broadband noise across the 3rd–6th harmonics after subtracting the fundamental harmonic components (3f0 ± 0.2f0, 4f0 ± 0.2f0, 5f0 ± 0.2f0, 6f0 ± 0.2f0). Analysis was performed in MATLAB. Based on PCD, 0.9 MPa was sufficient for ND activation.

Clot preparation: Bovine whole blood (acid citrate dextrose anticoagulated) was mixed with 2.75% CaCl2 at 10:1 (50 ml blood : 5 ml CaCl2). Unretracted clots were formed in plastic centrifuge tubes; retracted clots in borosilicate glass pipettes (per Sutton et al.). All clots incubated at 37 °C for 3 h, then stored at 4 °C for 2–16 days. For experiments, clots were standardized to 10 mm length, 5 mm diameter, and mass 150 ± 20 mg. Total tested: 54 retracted and 33 unretracted clots.

Treatment agents and conditions: Lipid-shelled decafluorobutane (DFB) microbubbles were synthesized and condensed to liquid-core NDs (average size 100–200 nm; stock ~10^10 ND/ml), then diluted to 10^8 ND/ml for treatment. MBs had mean diameter 1.1 µm; treatment concentration 10^8 MB/ml. tPA (Cathflo Activase) was prepared to 1000 µg/ml stock, stored at −20 °C, and diluted to 1.0 µg/ml working concentration in PBS. For combination groups, tPA was added to ND or MB solutions to 1.0 µg/ml.

Experimental flow model: An in vitro venous flow setup included a degassed water reservoir, a 37 °C water bath, and a waste container. System pressure was maintained at 3.5 ± 0.5 mmHg (DPGA-04 gauge). Flow rate was 50 ml/min. A mesh-supported inlet section held the clot to maintain partial occlusion. The FVI transducer with integrated injection lumen was positioned via the same inlet to insonate and deliver agents directly onto the clot.

Treatment groups and ultrasound settings for lysis: For retracted clots, groups included: control (PBS, no US), tPA alone (1.0 µg/ml, no US), tPA + US, MB + US (10^8 MB/ml), MB + tPA + US, ND + US (10^8 ND/ml), and ND + tPA + US. Sonication for lysis used PNP = 0.9 MPa at 700 kHz (other pulse parameters as per PCD unless noted). Statistical analysis employed one-way ANOVA with Tukey’s HSD; significance threshold p < 0.05. Sample size for retracted clot lysis groups: N = 6 per condition.

• Nanodroplet activation and cavitation (PCD):
  • Stable cavitation dose (V-Hz) increased with PNP: 0.52 ± 0.02 (0.3 MPa), 0.46 ± 0.03 (0.6 MPa), 0.76 ± 0.02 (0.9 MPa), 0.97 ± 0.03 (1.2 MPa). Significant increases at 0.9 and 1.2 MPa vs control (p < 0.05); 0.6 MPa approached significance (p = 0.057); 0.3 MPa not increased.
  • Inertial cavitation dose (V-Hz): 11.33 ± 1.07 (0.3 MPa), 11.38 ± 1.44 (0.6 MPa), 10.77 ± 0.67 (0.9 MPa), 13.22 ± 0.35 (1.2 MPa). Significant increases at 0.6 and 0.9 MPa vs control (p < 0.05). Overall results suggest stable cavitation as the primary operative regime.
  • A PNP of 0.9 MPa was sufficient for low-boiling point ND activation.
• Retracted clot lysis (percent mass decrease; N = 6 per group; PNP 0.9 MPa):
  • Control: 9 ± 8%
  • tPA alone: 9 ± 5%
  • tPA + US: 16 ± 5%
  • MB + US: 14 ± 9%
  • MB + tPA + US: 17 ± 9%
  • ND + US: 30 ± 8%
  • ND + tPA + US: 40 ± 9%
  • ND + tPA significantly outperformed tPA alone, tPA + US, MB + US, and MB + tPA + US. ND + US significantly outperformed control, tPA alone, and MB + US, and approached significance vs tPA + US (p = 0.07) and MB + tPA + US (p = 0.09).
• Efficacy achieved at 0.9 MPa (within FDA mechanical index safety limit), contrasting with external HIFU/histotripsy approaches that require much higher PNPs (up to ~15 MPa).

The study demonstrates that an intravascular forward-viewing transducer can activate low-boiling point nanodroplets at relatively low PNP (0.9 MPa) and, when combined with tPA, significantly enhances lysis of retracted clots compared with conventional MB-mediated or tPA-alone strategies. These findings address the challenge of treating dense, low-porosity retracted clots by leveraging ND penetration and cavitation to create microchannels, improving mechanical disruption and drug diffusion. The intravascular approach focuses acoustic energy directly on the clot, potentially improving efficiency versus transcutaneous methods that must transmit through intervening tissues. Compared to prior work indicating limited benefit of MB-mediated sonothrombolysis in retracted clots, ND + tPA provides a significant improvement while using acoustic outputs within safety limits. In unretracted clots, ND-mediated sonothrombolysis performs comparably to MB- and tPA-mediated methods, supporting its broader applicability.

This work presents the first use of low-boiling point, phase-change nanodroplets with an intravascular forward-viewing ultrasound transducer for sonothrombolysis of retracted clots. The FVI transducer effectively activated NDs at 0.9 MPa and, in combination with a low dose of tPA, achieved significantly greater lysis of retracted clots than MB- or tPA-based approaches alone. The approach operates within clinical safety limits and may overcome limitations of treating retracted thrombi that resist standard therapies. Future work should include comprehensive safety assessments (e.g., debris sizing, vessel injury), optimization of ultrasound parameters (duty cycle, pulse width, treatment duration), refinement of in vitro models to better mimic venous hemodynamics, and mechanistic studies of ND penetration and channel formation to further enhance efficacy.

Primary limitations include the use of an in vitro venous flow model that may not fully capture in vivo human physiology and vessel–clot interactions. Safety endpoints such as clot debris size distribution and endothelial or vessel wall damage were not assessed and require future study. Ultrasound parameter space was not fully optimized for ND-mediated lysis (e.g., duty cycle, pulse width, treatment duration). Mechanistic contributions beyond cavitation (e.g., ND penetration and microchannel formation) were hypothesized but not directly measured.