Intravascular sonothrombolysis device and system

The catheter size for PE treatments is about 6 F to 11 F (i.e. 2 to 3.7 mm in diameter)27, 28, so the custom catheters described herein composed of a miniaturized forward-looking transducer (lateral dimension <1.5 mm) and a microbubble injection tube (outer diameter of 1.1 mm), were developed as an 8F-prototype catheter (diameter of 2.7 mm). With this catheter device, sub-megahertz frequency ultrasound waves are excited to yield stable and inertial cavitation of locally-injected microbubbles near a target blood clot (Fig. 1(A)). The main thrombolysis mechanism is cavitation-induced microstreaming, which causes shear stress on the clot structure29, 30. Note that the present system excludes rt-PA injection, although it is likely that therapy using this system would involve low-dose use of thrombolytic drugs to avoid distal embolism caused by the fragmented clot particles. We anticipated that discernible improvement in sonothrombolysis efficiency without any thrombolytic agents can be a useful indicator of ultrasound-enhanced fibrinolysis by using our device since the accelerated efficiency arising from improved rt-PA penetration in MCA-involved therapy has been previously reported in detail16, 31. In order to attain sufficient pressure output for the bubble cavitation at sub-megahertz frequency, stacked-type piezoelectric transducers were designed and fabricated with the resonance frequency of 620 kHz (Fig. 1(B)). Although the low operating frequency is advantageous in yielding inertial cavitation of the MCA, the wide beam width due to the low frequency is inappropriate for precise insonation. Hence, we built a custom concave lens made of an aluminum oxide (Al 2 O 3 )/epoxy mixture for beam focusing to the target clot, minimizing the exposure area of the vessel wall. More detailed transducer design and fabrication procedures are provided in the Supplementary material (S1 and S2). The fabricated transducers with and without the concave lens were assembled with a microbubble-injection tube and 10 AWG-polyimide housing (outer diameter of 2.7 mm). The developed sonothrombolysis system is shown in Fig. 1(C). Thrombolysis efficiency of the developed system was evaluated in vitro. A bovine blood clot sample (200 mg ± 10%) was stored in a vessel mimicking tube (Fig. 1(D)), and the transducer was positioned close to the target clot (<0.5 mm from the aperture) using a 3-axis motion stage.

Figure 1 Microbubble-mediated intravascular sonothrombolysis system. (A) Principle of microbubble-mediated intravascular sonothrombolysis by using a catheter-tipped forward-looking transducer; low-frequency ultrasound waves generate stable and inertial cavitation of locally injected microbubbles near the target blood clot. (B) Customized 620 kHz stacked piezoelectric transducers integrated with a microbubble-injection tube and polyimide housing: concave- and planar-aperture (lateral dimension <1.5 mm) prototypes (scale bar = 5 mm). (C) In vitro experiment setup; a tygon tube (ID: 4 mm, OD: 5.6 mm) was used as a vessel-mimicking structure. The hydrophone was used to detect the microbubble response. (D) Bovine blood clot sample (200 mg ± 10%) located in the tygon tube with 50 μl saline water (scale bar = 5 mm). The figures (A) and (C) were drawn through the combined use of Solidworks Education Edition, Microsoft PowerPoint, and Adobe Illustrator CC. The figures (B) and (D) were captured using a digital SLR camera (Canon EOS Rebel T3) with 50 mm lens (Canon, f/2.5 Compact Macro Lens), cropped and aligned using Microsoft PowerPoint and Adobe Illustrator CC. Full size image

Characterization of the prototype transducers

The prototype transducers were acoustically characterized to confirm that the acoustic pressure is sufficient for cavitation of the injected microbubbles in the confined field of insonation. Beam profiles of both planar and concave aperture-prototypes are shown in Fig. 2(A). −6 dB beam diameters of planar- and concave-aperture prototypes are 3.1 mm and 1.3 mm, respectively. We confirmed that the concave lens is able to confine the beam width despite the small aperture area in terms of wavelength. Although theoretical design guidelines for the focusing lens recommend large aperture diameters (>5λ), in practicality, lenses can be designed with rather modest aperture/wavelength ratios32. Due to the small aperture, the measured beam, which was designed to have a 1 mm focus based on the geometry of concave lens, exhibited a diffuse focal region rather than a single focal distance. However, the focused aperture showed a spatially confined beam compared to the planar transducer. In order to evaluate the transmitting sensitivity, the measured pressure output (1 mm away from the aperture) of both planar and concave transducers were compared for various voltage inputs as shown in Fig. 2(B). Voltage inputs were varied in the range of 10–80 V pp . The concave aperture prototype exhibited approximately 2.5-fold higher peak-to-peak pressure (PTP) and 2.3-fold higher peak-negative-pressure (PNP) outputs than the planar aperture prototype. One quantitative metric related to microbubble-mediated sonothrombolysis is mechanical index (MI), which is defined as PNP (in MPa) divided by the square root of the operating frequency (in MHz). Within the voltage input range, the maximum MI with planar and concave aperture design were 0.4 and 1.0, respectively. For clinical imaging, microbubble contrast agents are approved for use in humans at MIs up to 0.8 (Definity, Lantheus Medical Imaging, North Billerica, Massachusetts, formerly Bristol-Myers Squibb Medical Imaging), though they are often flashed with short bursts at higher MI in destruction-reperfusion imaging studies33,34,35. Mechanical indices which exceed this imaging limit have been reported in sonothrombolysis36,37,38. It was previously reported that the approximate MI threshold for MCA cavitation is 0.3539 when the excitation frequency is lower than microbubble resonance frequency (1–5 MHz for MCA with an approximated microbubble diameter of 1 μm), and we confirmed in this work that both prototypes with different aperture designs are able to yield inertial cavitation during microbubble-mediated thrombolysis therapies.

Figure 2 Acoustic characterization of customized stacked-type intravascular ultrasound transducer. (A) Measured beam profiles of planar- (A1) and concave-aperture (A2) prototypes; the red arrows indicate the radiation direction, and the red contour lines denote the mechanical index of 0.3 with voltage input of 80 V pp which implies the approximated threshold of inertial cavitation of microbubbles. −6 dB beam diameters of planar- and concave-aperture prototypes are 3.1 mm and 1.3 mm, respectively. (B) Measured pressure output (1 mm away from the aperture) with voltage inputs of 10–80 V pp (n = 2). The concave aperture prototype exhibits approximately 2.5-fold increase in peak-to-peak pressure (PTP) and 2.3-fold increased peak-negative-pressure (PNP) relative to the planar aperture prototype. (*P < 0.05). Full size image

In vitro tests

We investigated the reduction in volume of the bovine blood clot using the developed sonothrombolysis system with the planar lens prototype transducer. Initial in vitro tests were conducted using a 120 mg clot positioned in a tygon tube (Fig. 3(A)). The captured images with 10 min-interval show gradual volume reduction. The 40 min treatment includes a 30 sec break for every 5 min of sonication in order to allow new microbubbles penetrate into the fibrin clot40. After a 40 min treatment, the target clot size reduced to about 35% of its original size (mass reduction from 120 mg to 50 mg, Fig. 3(B)). The position of the transducer was controlled to keep the face of the transducer less than 0.5 mm from the clot surface. Since relatively high concentrations of MCA (508 bubbles/ml) were used in this initial test, we observed some groups of undestroyed microbubbles attached to the transducer and the inner surface of the tygon tube during the treatment even though the output pressure with the 80 V pp input (PNP of 250 kPa) is sufficient to destroy the bubbles. This remaining population may include microbubbles which have coalesced into larger microbubbles41. Observed remaining bubbles may also be due to the smaller cavitation zone compared to the inner diameter of the vessel-mimicking tube and the period during which bubbles were injected without insonation. Despite the very low (100 μl/min) bubble infusion rate, intact bubbles can be spread around the vessel wall during 30 sec infusion without insonation. During treatment with a 10% duty cycle, the temperature at the clot boundary (insonation area) remained unchanged as measured by calibrated thermocouple (37 °C). This result indicates that the clot was dissolved by cavitation-induced microstreaming without measurable ultrasound-induced thermal effects.

Figure 3 In vitro clot lysis by microbubble-mediated intravascular ultrasound. (A) Captured images during 40 min treatment by using the planar-aperture prototype (input conditions of 620 kHz, 80 V pp , 10% duty cycle with 100 μl/min injection of 508 bubbles/ml). A smaller (120 mg) clot was used for monitoring the volume reduction (scale bar = 3 mm). Due to the high bubble concentration (508 bubbles/ml) we observed that the undestroyed bubbles were attached to the transducer and the inner surface of the tygon tube. (B) A 120 mg clot was reduced to 50 mg after the 40 min treatment (scale bar = 3 mm). Full size image

We then conducted further in vitro tests with various input parameters. The two different designs of prototype transducers (planar and concave apertures) were used, and the lysis results were compared. With the same input conditions of voltage, duty cycle, microbubble concentration, and microbubble injection rate, the results obtained by the planar transducer and the concave transducer showed an average lytic rate of 0.7%/min and 0.67%/min, respectively. Interestingly, despite the much higher pressure output (>100% higher at the focal point) of the concave transducer, both prototypes realized similar (<5% difference) mass reduction (Fig. 4(A)). Although the confined beam has higher pressure at the cavitation zone, which is determined by spatial volume within the ultrasound beam where the MI is higher than ~0.3, thrombolysis rate of the planar transducer is similar to that of concave transducer, as shown in the Fig. 2(A). Thus, the planar transducer was used for further experiments while varying other parameters. Since we observed from the initial test that intact microbubbles exist when the bubble concentration is too high, we investigated effect of MCA concentration to find the appropriate MCA injection condition (Fig. 4(B)). The percent mass loss increases with the bubble concentration. This is because larger amount of MCA cavitation can generate larger exposure area of microstreaming-induced shear force29, 30, 42. We also found that the higher percent mass loss was achieved with the higher voltage inputs and higher duty cycle (Fig. 4(C) and (D)). The in vitro test results with various input parameters had a similar tendency with the previously reported sonothrombolysis results obtained by using a commercially available diagnostic 2.5-MHz transducer24.

Figure 4 In vitro test results with different input parameter. (A) Mass reduction of the clots (200 mg ± 10%) in accordance with treatment time of 15–60 min (620 kHz operating frequency, 80 V pp input voltage, 10% duty cycle, 100 μl/min injection of 107 bubbles/ml). Despite of the different pressure output (>100% difference) both aperture prototypes realize similar (<5% difference) mass reduction; the difference of mass reduction showed no statistical significance. (B–D) Mass reduction upon variation of treatment condition; fixed parameters for each test are 30 min treatment time, 80 V pp input voltage, 10% duty cycle (5 ms burst duration and 305 cycle-burst), 107 bubbles/ml injection with 100 μl/min flow rate (n = 3). (B) Variation of microbubble concentration. (C) Variation of input voltage. (D) Variation of duty cycle. (*P < 0.05, **P < 0.01). Full size image

Next, we investigated the required treatment time to reach 100% mass reduction. The two designs of prototyped transducers were used in these tests. The operating parameters used were determined based on the previous parameter study results presented in Fig. 4(A). First, we observed that the complete thrombolysis was not achieved with only microbubble-mediated sonothrombolysis treatment. After about 3.5 h treatment, red cells inside the clot were almost entirely lysed but some of the fibrin structure remained. Even with a treatment time longer than 4.5 h, the fibrin network was not completely removed (Fig. 5(B)). Hence, further tests were conducted until 90% mass reduction, and the required treatment time was analyzed. As we found in the previous in vitro test results, the planar and concave transducers showed similar (approximately 5.2% difference) treatment time for 90% mass reduction. This was expected due to the similar cavitation volume (Fig. 2(A)) of both the planar and concave transducers. Since the fibrin fibers are cleaved by the active enzyme plasmin43, 44, we hypothesize that 100% thrombolysis might be expected with a local administration of a minimum amount of thrombolytic agent. Regardless of the improved penetration of rt-PA to the clot, we confirmed that the microbubble-mediated intravascular sonothrombolysis by using our miniaturized transducers can realize a lytic rate of 0.7 ± 0.15%/min.