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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Ultrasound Med Biol. 2021 Aug 23;47(11):3231–3239. doi: 10.1016/j.ultrasmedbio.2021.07.018

Safety Evaluation of a Forward Viewing Intravascular Transducer for Sonothrombolysis: an In-Vitro and Ex-Vivo Study

Leela Goel a,b, Huaiyu Wu a, Bohua Zhang a, Jinwook Kim b, Paul A Dayton b, Zhen Xu c, Xiaoning Jiang a,*
PMCID: PMC8487993  NIHMSID: NIHMS1728874  PMID: 34446331

Abstract

Recent in-vitro work has demonstrated that a forward-viewing intravascular (FVI) transducer can be applied for sonothrombolysis applications. However, the safety of this device has yet to be evaluated. In this study, we evaluated the safety of this device in terms of tissue heating, vessel damage, and particle debris size during sonothrombolysis using microbubbles or nanodroplets with tissue plasminogen activator (tPA), in both retracted and unretracted blood clots. The in-vitro and ex-vivo sonothrombolysis tests using FVI transducers showed temperature rise of less than 1°C, no vessel damage as assessed by histology, and no downstream clot particles greater than 500μm. These in vitro and ex-vivo results indicate that the FVI transducer poses minimal risk for sonothrombolysis applications, and should be further evaluated in animal models.

Keywords: intravascular sonothrombolysis, safety, retracted clots, nanodroplets, microbubbles, ultrasound assisted thrombolysis, tissue plasminogen activator

Introduction

Our group has demonstrated that a forward-viewing intravascular transducer can be used to improve the sonothrombolysis efficiency for both retracted an unretracted blood clots, in vitro (Kim et al., 2017; Goel et al., 2020, 2021; Zhang et al., 2019). This transducer was developed to improve the sonothrombolysis rate with a low of dose of tPA (1.0μg/ml), microbubbles, and nanodroplets. We have also optimized the localized concentration of microbubbles and examined the dynamics of nanodroplet and microbubble enhancement of ultrasonic clot lysis. (Kim et al., 2020, 2017; Goel et al., 2020). Given these promising initial in vitro sonothrombolysis results, it is important to study the safety of this device before being applied to in vivo animal or human studies.

Before using the forward viewing intravascular transducer for in vivo animal or human trials for sonothrombolysis, it is critical to evaluate the safety risk of the device. Previous clinical trials for transcranial sonothrombolysis have demonstrated that there may be an increased risk of intracranial hemorrhage due to damage in the middle cerebral artery compared to standard treatments for middle cerebral artery occlusions (Daffertshofer et al., 2005; Alexandrov et al., 2004, 2019). While the EKOS intravascular device has been applied for intravscular sonothrombolysis treatment clinically, to our knowledge there have been only a few studies examining the safety of catheter directed sonothrombolysis and only one specifically studying the EKOS transducer (Grommes et al., 2011; Tachibana and Tachibana, 1997). As such, there is a need for pre-clinical studies which directly study the safety of intravascular sonothrombolysis techniques.

The primary safety concerns with sonothrombolysis are excessive tissue heating, formation of large clot particles, and endothelial damage of the blood vessels (Fry, 1979; Abramowicz et al., 2008; Chueh et al., 2013). Excessive tissue heating may cause localized inflammation or cell damage, which can further exacerbate a pathological coagulation pathway and induce more injury. Excessively large clot particles which may form after clot dissolution can increase the risk for blocking downstream vessels, and in the worst case cause a pulmonary embolism or stroke (Chueh et al., 2013). Finally, damage to blood vessels may cause internal hemorrhage in the patient, as well as exacerbate the coagulation pathway resulting in the formation of new blood clots. Current clinical catheter directed treatments for blood clots include mechanical thrombectomy, which can result in particle sizes of over 1mm in diameter to form hazardous emboli and may also cause endothelial damage themselves due to mechanical perturbations or punctures (Chueh et al., 2013; Muller-Hulsbeck et al., 2002, 2001; Sharafuddin and Hicks, 1998).

Given the large variety of sonothrombolysis techniques which can be used with the same ultrasound device, (i.e. thrombolytic drug/tissue plasminogen activator (tPA) mediated, microbubble, and nanodroplet mediated techniques), a systematic pre-clinical safety study is needed to directly compare the safety profiles of different sonothrombolysis techniques with the same device (Goel and Jiang, 2020). While safety is of paramount concern when developing a new therapeutic ultrasound technique, there have been few pre-clinical systematic studies evaluating the safety of sonothrombolysis techniques. Most studies have examined clot debris formation and vessel damage when using high intensity focused ultrasound (HIFU), low intensity focused ultrasound (LIFU), or histotripsy based technologies (Guo et al., 2019; Zhang et al., 2017, 2015b,a; Maxwell et al., 2009, 2011; Burgess et al., 2012; Wright et al., 2012; Sakharov et al., 2000; Zhong et al., 2019). A few studies from the late 1990s and early 2000s quantified the temperature changes with sonothrombolysis using a variety of ultrasound devices (Atar et al., 2001; Tachibana and Tachibana, 1997; Sakharov et al., 2000; Riggs et al., 1997; Francis et al., 1992; Kashyap et al., 1994; Zhong et al., 2019). A direct comparison of the primary safety parameters of interest for different sonothrombolysis approaches using the same device would aid in understanding the benefits and risks of these techniques.

In this study we aim to evaluate the safety of our forward viewing intravascular transducer, in-vitro and ex-vivo, for tPA and contrast agent mediated sonothrombolysis. The temperature increase in tissue near the transducer interface, clot particle size generated by the sonothrombolysis process, and vessel damage caused by direct insonation of the ultrasound transducer were studied.

Materials and Methods

Ultrasound Parameters

For all experiments, a 7-layer stacked transducer was used and prepared in a method similar as previous studies (Kim et al., 2017; Goel et al., 2021). Briefly, the transducer was composed of 7 PZT-5A plates with an Al2O3/epoxy matching layer. The final aperture size was 1.5 mm, with an axial −6 dB acoustic field length of approximately 2mm. The transducer center frequency was 750 kHz and the −6dB fractional bandwidth of 40%, impedance of 225Ω, at 747 kHz, and capacitance of 1.43 nF at 1kHz. The peak to peak driving voltage of the transducer was 50V to achieve a peak negative pressure output of 0.9 MPa.

The excitation frequency used was 700 kHz with a 5 ms pulse length and 5.7% duty cycle. The peak negative pressure output was 0.9 MPa and the total treatment duration was 30 minutes for each group of tests. The mechanical index (MI) with these driving conditions 1.1. These parameters were chosen based on previous studies which demonstrated effective sonothrombolysis outcomes with these measures (Goel et al., 2020, 2021; Kim et al., 2017). The transducer was integrated into a catheter system with a single lumen for treatment injection.

Treatment Conditions

Treatment conditions were chosen based on previous studies (Kim et al., 2017; Goel et al., 2020, 2021). Tissue plasminogen activator (tPA) was prepared per the manufacturer’s instructions as described previously (Goel et al., 2020, 2021). tPA was diluted to a working concentration of 1.0μg/ml for treatments. Microbubbles and nanodroplets were prepared as described previously, (Sheeran et al., 2012; Kim et al., 2020). Custom lipid-shelled, decafluorobutane (DFB) microbubbles (MB) were prepared to a final working concentration of 108 MB/ml. Similarly, custom low boiling point nanodroplets (ND) were prepared to a final concentration of 108 ND/ml. In combined MB + tPA and ND + tPA conditions, the tPA concentration remained at 1.0μg/ml, MB concentration of 108 MB/ml, and ND concentration of 108 ND/ml. Phosphate buffered saline (PBS) was used for the no ultrasound and ultrasound (US) alone conditions. Treatments were injected through the catheter lumen within 0.5mm of the clot surface via syringe pump at a rate of 0.100 mL/min. The treatment solutions were injected directly onto the location of interest. As established from previous studies, the ultrasound output was manually switched “on” for 2 minutes and “off” for 30 seconds throughout the 30 minute treatment period to allow for penetration of the treatment solutions into the clot (5.7% duty cycle) (Kim et al., 2017; Goel et al., 2020, 2021).

Heating

In order to estimate the potential maximum heating outcomes, experiments were conducted in a static, non-flow model, similar to that of a previous study (Goel et al., 2020). Additionally, heating experiments were conducted in chicken breast tissue to allow for maximal heat energy to be retained in the tissue and not be dissipated due to blood clot dissolution.

Chicken breast was placed and sealed into a tygon tube, and filled with PBS (Figure 1)). This tube was then placed in a 37°C water bath. A thermocouple (Fluke 179 True RMS Multimeter, Everett, WA, USA) was placed inside of the tissue within 1 mm of the transducer face and used to measure the temperature. The experimental conditions tested were US alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US. Temperature was measured and recorded for 30 minutes. The temperature peaks throughout the treatment were recorded and the maximum average temperature rise was calculated. Each condition was repeated 3 times (N = 18).

Figure 1:

Figure 1:

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Diagram of experimental setup for tissue heating experiment.

One-way analysis of variance (ANOVA) was performed in order to assess statistically significant differences p < 0.05.

Vessel Damage

Bovine leg veins were obtained from a commercial animal tissue supplier (Sierra for Medical Science, Whittier, CA, USA) and did not require institutional animal care and use committee (IACUC) approval. Bovine leg veins were stored in cold phosphate buffered saline (PBS) before use. Vessels were cleaned and trimmed to approximately 1 cm in length (Figure 2). Vessels were then spread open and mounted and the inside of the vessel lumen was treated with either PBS alone (control), tPA alone, US alone, tPA + US, MB + US, MB + tPA + US, ND + US, or ND + tPA + US. The internal lumen of the vessel was directly treated in a single location for the entire treatment duration of 30 minutes using the standard insonation scheme. Each condition was repeated 3 times. The treated vessels were then trimmed to the region of excitation and fixed in formalin for histology.

Figure 2:

Figure 2:

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Preparation of blood vessels. a) Whole bovine vessel, b) Prepared and mounted vessel, c) Ultrasound treatment of vessel.

Hematoxylin and eosin (H&E) staining was performed on the treated vessels. Three mirrored cross-sections were obtained for each sample at 0μm, 100μm, and 200μm, resulting in 6 sections per sample. Slides were digitally scanned at 10X and 20X magnification using an EVOS Fl Auto (EVOS FL Auto Imaging System, Life Technologies Corporation, Carlsbad, CA, USA). Approximately 300 images were evaluated in order to assess vessel damage.

Clot Particle Formation

Bovine blood clots were prepared as described previously (Goel et al., 2020, 2021). Bovine whole blood was mixed with calcium chloride in a 10:1 ratio (50 ml blood/5 ml calcium chloride). For retracted clots, the blood mixture was transferred to borosilicate glass pipettes and sealed. For unretracted clots, the blood mixture was transferred to plastic microcentrifuge tubes. After transfer to appropriate tubes, both unretracted clots and retracted clots were incubated in the same manner. The transferred mixture was then incubated in a 37°C water bath for 3 hours and then stored in a 4°C refrigerator for at least 2 days. The final clot ages were 2–16 days old. For each experiment, clots were prepared to a final mass of 150 ± 20mg. Each experimental condition was repeated 3 times each, for a total of 24 unretracted clots and 24 retracted clots.

Experiments were conducted in a venous flow model, similar to previous studies (Figure 3) (Goel et al., 2021). The flow model consisted of a degassed water reservoir, 37°C water bath (Poly ProBath, Revolutionary Science, Shafer, MN, USA), an inlet for the transducer/catheter system, and a passive filtration system. The pressure was maintained at 3.5 ± 0.5 mmHg and monitored with a digital pressure gauge (DPGA-04, Dwyer Instruments, Inc., Michigan City, IN, USA) with a flow rate of 50 mL/min. Both retracted and unretracted clots were treated with PBS alone (control), tPA alone, ultrasound only, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US for 30 minutes. The percent mass decrease of the clots before and after treatment were calculated as the metric for clot lysis.

Figure 3:

Figure 3:

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Diagram of experimental setup for clot particle measurement.

Using the passive filtration system, clot particles were serially filtered with nylon plastic meshes (9318T44, 9318T26, 9318T21m, 9318T48, McMasterCarr, Elmhurst, Illinois) with mesh sizes of 500μm, 200μm, 100μm, and 50μm. Each filter was then dried for at least 24 hours. A microscope was used to examine the filters for clot debris. Photographs of the debris were captured and particle size was estimated using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA). The total number of particles which were larger than 50μm were recorded, as well as the distribution of the size of the particles > 50μm for each experimental condition.

One-way ANOVA was performed in order to assess statistically significant differences in clot particle size and distributions, with a significance level of p < 0.05. Linear regression was performed in order to examine the relationship between percent mass decrease and total number of clot particles observed.

Results

Heating

The average temperature rise were 0.39 ± 0.09°C, 0.64 ± 0.08°C, 0.48 ± 0.10°C, 0.46 ± 0.08°C, 0.75 ± 0.08°C, and 0.60 ± 0.07°C for the US alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups respectively (Figure 4b). A sample temperature curve is shown in Figure 4a. There was no statistical difference in temperature rise between the MB + US and MB + tPA + US groups, or the tPA + US and ND + tPA + US groups. The average temperature rise was less than 1°C for all conditions, which was not sufficient to cause biological damage (Fry, 1979; Abramowicz et al., 2008).

Figure 4:

Figure 4:

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a) Example temperature rise curve for a microbubble + tPA + US sample. b) Average temperature rise for each experimental condition with ultrasound. *,†,‡,•, and Δ indicates significance compared to US alone, tPA + US, MB + US, MB + tPA + US, and ND + US respectively (p < 0.05).

Vessel Damage

Gross morphology showed no macroscopic damage for all vessel samples. Histology from each sample indicated there were no observed changes in endothelial cell orientation or spacing along the vessels or between treatment condition. (Figure 5).

Figure 5:

Figure 5:

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Representative images of H&E staining of bovine veins to evaluate vessel damage after treatment conditions for a–b) control group, c–d) MB + tPA alone, and e–f) ND + tPA + Ultrasound. Scale bar indicates 200μm.

Clot Particle Formation

In retracted clots, the average total number of clot particles released from each treatment was ranged from 0 to 22 (Figure 6). The largest number of particles observed was 69 for one sample in the MB + tPA + US condition. There were no significant differences in the distribution of particle sizes between treatment conditions. Only one particle > 300μm was observed in the MB + US condition, with a size of 409μm. In unretracted clots, the average total number of clot particles released from each treatment was ranged from 6 to 18 (Figure 7). The largest particle counts observed were 30 for one sample in the ND + tPA + US group; 28 for one sample in the ND + US; 27 for one sample in the MB + US; and 36 for one sample in the tPA alone conditions. There were no significant differences in the total number of particles or the distribution of particle sizes between treatment conditions. Two particles > 300μm was observed in the ND + US condition, with a size of 346μm and 306μm; one particle in the MB + tPA + US condition, with a size of 362μm; and one particle in the US alone condition, with a size of 356μm.

Figure 6:

Figure 6:

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a) Average total number of particles > 50μm measured and b) Distribution of particle sizes in retracted clots. * indicates p < 0.05. There were no statistically significant findings in the debris distribution between treatment conditions.

Figure 7:

Figure 7:

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a) Average total number of particles > 50μm measured and b) Distribution of particle sizes in unretracted clots. There were no statistically significant findings in the total number of particles or the debris distribution between treatment conditions.

There was no correlation between the percent mass decrease of the clots and the total number of particles formed, with an R2 = 0.02 (Figure 8). In the retracted clots, the average percent mass decrease was 4 ± 2% for the control group, 17 ± 9% for tPA alone, 14 ± 4% for US alone, 24 ± 12% for tPA + US, 29 ± 1% for MB + US, 27 ± 6% for MB + tPA + US, 26 ± 6% for ND + US, and 36 ± 6% for ND + tPA + US. In the unretracted clots, the average percent mass decrease was 50 ± 6% for the control group, 45 ± 10% for tPA alone, 54 ± 7% for US alone, 60 ± 2% for tPA + US, 67 ± 5% for MB + US, 72 ± 9% for MB + tPA + US, 67 ± 5% for ND + US, and 73 ± 5% for ND + tPA + US.

Figure 8:

Figure 8:

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Percent mass decrease vs. total number of clot debris > 50μm for both retracted and unretracted clots. There was no correlation between percent mass decrease and the total number of clot particles formed R2 = 0.02

Discussion

In this study, we investigated the safety of the forward-viewing intravascular transducer for sonothrombolysis applications, in vitro. After a 30 minute treatment, we found a maximum tissue temperature rise of less than 1°C for all conditions, no evidence of ex vivo vessel wall damage, and no clot particles > 500μm formed, providing initial evidence on the safety of this device.

Our heating results correspond well with previous sonothrombolysis studies examining these ultrasound parameters. One previous study by Tachibana et al examining the temperature rise with a prototype intravascular transducer also found a temperature rise of less than 1°C for all conditions tested (Tachibana and Tachibana, 1997), which corresponds well to the results in this study. Other studies utilizing HIFU, Doppler Ultrasound, and low intensity focused ultrasound that used higher pressures and/or higher PRFs found temperature increases of up to 9°C (Sakharov et al., 2000; Riggs et al., 1997; Francis et al., 1992; Zhong et al., 2019; Kashyap et al., 1994). Given that our heating tests were conducted in a static model, we would expect in vivo temperature rises to be even less.

Our study revealed no observable damage when the inside of the vessel lumen was directly exposed to the FVI transducer. Other in vivo animal studies using the EKOS intravascular transducer, a custom intravascular transducer, HIFU, histotripsy, LIFU, and Doppler Ultrasound found minimal or no endothelial damage for the blood vessels examined (Grommes et al., 2011; Zhang et al., 2017; Maxwell et al., 2009, 2011; Burgess et al., 2012; Shimizu et al., 2012; Tachibana and Tachibana, 1997; Riggs et al., 1997; Kashyap et al., 1994; Zhong et al., 2019). Given that for HIFU and histotripsy, the peak negative pressures are much higher than the peak negative pressures used in this study with the FVI transducer (up to 15 MPa vs. 0.9 MPa), we would expect our FVI transducer to also have minimal vessel damage in a in vivo setting. Additionally, for our vessel damage study, the FVI transducer was directly insonating the interior lumen. In an in vivo case, the transducer would be directly insonating the clot and not the vessel, therefore we would predict an even smaller risk for vessel damage to occur in an in vivo case. However, one major limitation of this ex-vivo analysis is the inability to assess inflammation or hemorrhage due to the excitation. While this study was able to identify vessel perforation, destruction, and gross morphological changes, in vivo animal studies are necessary to evaluate non-mechanical damage to the vessel.

The largest clot particle size from our treatment was below 500μm. The particle size analysis from our FVI transducer matches well with a study by Zhang et al. using a microtripsy approach (Zhang et al., 2015a)). In that study, they found very few particles > 300μm and < 20 particles greater than 100μm regardless of the ultrasound parameters and efficacy of the treatment, which matches well with our findings that the total number of large particles generated are independent of the mass decrease of the clots (Zhang et al., 2015a). In terms of particle size distribution, the researchers also found similar distributions across treatment conditions (Zhang et al., 2015a). Other in vivo studies found no evidence of a pulmonary embolus from sonothrombolysis techniques (Grommes et al., 2011; Zhang et al., 2017; Maxwell et al., 2011). While these initial in vitro studies indicate a low risk of dangerous emboli formation, further clot particle analysis and in vivo animal testing is warranted.

It should be noted that the percent mass decreases observed in this study are larger than those observed in our previous study, particularly for unretracted clots (Goel et al., 2021). However, these differences may be attributed to variability in the blood supply. Additionally, the trends in clot mass decrease and treatment condition still hold as compared to previous literature (Goel et al., 2020; Sutton et al., 2013). While only heating, vessel damage, and large clot particle formation were considered in this study, other potential concerns such as hemolysis or other negative physiological changes should be observed in pre-clinical studies to fully understand the safety of this device.

When choosing the appropriate sonothrombolysis approach, safety and efficacy must be considered. From these data, we see that in retracted clots, nanodroplet mediated techniques generate fewer clot particles and smaller sized particles compare to microbubble mediated techniques. Additionally, ND + tPA + US provides an improvement in efficacy compared to the MB + tPA + US condition. But in unretracted clots, microbubble and nanodroplet mediated techniques have similar safety and efficacy profiles. In that case, the risk assessment of the relative benefits and risks of using tPA can be considered versus using a contrast agent mediated technique with ultrasound in unretracted clots. In vivo animal studies will be required in order for better risk analysis of the efficacy versus safety of different sonothrombolysis approaches. The appropriate type of sonothrombolysis treatment (microbubbles, nanodroplets, with or without thrombolytic drugs) may differ for the clot type being treated and underlying patient conditions.

Conclusion

In this study, we evaluate the safety of the forward-viewing intravascular transducer for sonothrombolysis, in vitro and ex vivo. We examine tPA, microbubble, and nanodroplet mediated sonothrombolysis in both retracted and unretracted clot. We found that this transducer poses minimal risk of excessive tissue heating, vessel damage, or hazardous embolization, similar to that found in other established sonothrombolysis techniques. We plan to confirm the results in future in vivo safety testing.

Acknowledgments

This work was supported by National Institutes of Health grant R01HL141967. The authors acknowledge Sonovascular, Inc. for fabrication of the integrated catheter system. The authors also thank Brian Velasco for preparation of the ultrasound contrast agents and Matthew Blizzard for filter preparation.

Footnotes

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Conflict of Interests

Xiaoning Jiang has financial interest in Sonovascular, Inc. who licensed an intravascular sonothrombolysis technology from NC State. Paul Dayton is an inventor on several patents describing the low-boiling point phase change nanodroplets described here, and is a co-founder of Triangle Biotechnology, Inc., which has licensed these patents. Zhen Xu has financial interest in HistoSonics.

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