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Published in final edited form as: Phys Med Biol. 2023 Feb 21;68(5):10.1088/1361-6560/acb9d1. doi: 10.1088/1361-6560/acb9d1

A feasibility study on removing lipid deposition in atherosclerotic plaques with ultrasound-assisted laser ablation

Rohit Singh 1, Koji C Ebersole 2, Xinmai Yang 1
PMCID: PMC11425588  NIHMSID: NIHMS2022411  PMID: 36804803

Abstract

Objective.

Atherosclerosis is the buildup of fats, cholesterol, and other substances on the inner walls of arteries. It can affect arteries of heart, brain, arms, legs, pelvis and kidney, resulting in ischemic heart disease, carotid artery disease, peripheral artery disease and chronic kidney disease. Laser-based treatment techniques like laser atherectomy can be used to treat many common atherosclerostic diseases. However, the use of laser-based treatment remains limited due to the high risk of complications and low efficiency in removing atherosclerostic plaques as compared with other treatment methods. In this study, we developed a technology that used high intensity focused ultrasound to assist laser treatment in the removal of the lipid core of atherosclerotic plaques.

Approach.

The fundamental mechanism to disrupt atherosclerostic plaque was to enhance the mechanical effect of cavitation during laser/ultrasound therapy. To promote cavitation, spatiotemporally synchronized ultrasound bursts of 2% duty cycle at 0.5 MHz and nanosecond laser pulses at 532 nm wavelength were used. Experiments were first performed on pig belly fat samples to titrate ultrasound and laser parameters. Then, experiments were conducted on human plaque samples, where the lipid depositions of the plaques were targeted.

Main results.

Our results showed that fat tissue could be removed with an ultrasound peak negative pressure (PNP) of 2.45 MPa and a laser radiant exposure as low as 3.2 mJ mm−2. The lipid depositions on the atherosclerostic plaques were removed with laser radiant exposure of 16 mJ mm−2 in synchronizing with an ultrasound PNP of 5.4 MPa. During all the experiments, laser-only and ultrasound-only control treatments at the same energy levels were not effective in removing the lipid.

Significance.

The results demonstrated that the addition of ultrasound could effectively reduce the needed laser power for atherosclerotic plaque removal, which will potentially improve treatment safety and efficiency of current laser therapies.

Keywords: atherosclerosis, excimer laser coronary angioplasty, ultrasound, cavitation

1. Introduction

Atherosclerosis is a medical condition in which arteries harden and narrow due to the buildup of fat, cholesterol, calcium and other substances on the inner artery walls. Atherosclerosis is the major cause of cardiovascular disease such as ischemic heart disease and ischemic stroke, and atherosclerotic cardiovascular disease (ACD) is a major cause of death in the United States (US) and worldwide. In 2019, ACD affected 523 million people worldwide and caused 18.6 million deaths (Roth et al 2020). In the US, ACD caused 697,000 fatalities in 2020 alone (Tsao et al 2022). The direct and indirect cost of ACD in 2017–2018 was $229 billion in the US (Medical Expenditure Panel Survey MEPS 2021).

Several medications can slow down or reverse the process of atherosclerosis. Cholesterol lowering medications, blood thinners and blood pressure medications are the most commonly used. Cholesterol lowering medications such as statins slow down the buildup of lipids in the arteries by reducing low-density lipoprotein cholesterol in the blood (Pinal-Fernandez et al 2018). Blood thinners such as aspirin are often used to prevent the formation of blood clots (Leys et al 2009). Blood pressure medications are used to reduce the risk of plaque rupture by controlling high blood pressure. If medications are not effective in treating the atherosclerotic plaque blockages, interventional surgeries will be performed on the affected portion of the arteries. The most common surgical procedure for coronary atherosclerosis is percutaneous coronary intervention (PCI), also known as angioplasty and stenting (Hoole and Bambrough 2020). In case of atherosclerosis in the carotid artery, the plaque buildup is more often removed surgically by a procedure known as endarterectomy (Rerkasem et al 2020), which is more effective than carotid angioplasty and stenting (Perkins et al 2010). For severe atherosclerosis in coronary arteries, also known as multi-vessel disease, a coronary artery bypass grafting can be used to create a bypass and redirect the blood flow around the blocked artery (Sipahi et al 2014).

Since the 1990s, laser technology has been used to modify atherosclerotic plaques during PCI, a technique commonly known as excimer laser coronary angioplasty (ELCA) (Cook et al 1991). This technique uses a nanosecond (125–200 ns) pulsed laser in the ultraviolet range (100–400 nm) to remove plaques by photochemical (Litvack et al 1988), photothermal (van Leeuwen et al 1993) and photomechanical (Topaz 1993) mechanisms. ELCA uses a high laser fluence in the range of 30–80 mJ mm−2 and a pulse repetition frequency of 25–80 Hz (Egred and Brilakis 2020, Tsutsui et al 2021). However, the use of high laser fluence and pulse repetition frequency results in complications (Ghazzal et al 1992, Litvack et al 1994). In early clinical trials, the efficacy of ELCA was less or almost the same as balloon angioplasty, but it increased the risk of vessel dissection and perforation (Litvack et al 1994, Appelman et al 1996, Holmes et al 1997, Köster et al 2000). As a result, the overall use of ELCA remains low due to the higher risk of complications as compared with non-ELCA PCI procedures (Sintek et al 2021).

In this study, we tested a new technique to disrupt atherosclerotic plaques. This new technique used combined ultrasound and laser irradiation to modify the plaque by enhancing cavitation. This technique is based on our previous technique, namely ultrasound-assisted endovascular laser thrombolysis (USELT), which removes blood clots using combined ultrasound and laser (Jo et al 2021, Singh et al 2021). A recent study has demonstrated that USELT is highly effective in dissolving blood clots in veins in an in vivo rabbit model (Singh et al 2021). The combination of ultrasound and laser results in enhanced cavitation (Li et al 2018, Singh et al 2020), which precisely disrupts and breaks the blood clot without causing any damage to the nearby tissues. In the current study, we further tested the feasibility of using combined ultrasound and laser to disrupt lipid-rich regions in atherosclerotic plaques. We focused our treatment on the soft lipid-rich mass in the plaque because the lipid-rich mass in plaque is the most dangerous component in the plaque, often resulting in atherothrombosis and strokes (Badimon and Vilahur 2014). We first titrated laser and ultrasound parameters with experiments on the lipid or fat portion of pork belly. Then experiments were conducted on human carotid plaque samples acquired during carotid endarterectomy surgery. Pork belly fat was used for the titration of experiment parameters due to its similar composition to the lipid/fat in atherosclerotic plaques (Soladoye et al 2015). The results from the experiments on pork belly fat were used to select ultrasound and laser parameters for the experiment on carotid plaques. The major goal of this study is to demonstrate the feasibility of combined laser and ultrasound therapy for disrupting atherosclerotic plaques at a relatively low laser-power level, which could improve the safety of laser-based technology for atherosclerotic plaque modification.

2. Methods

2.1. Experimental setup

A detailed schematic of the experimental setup is shown in figure 1. The setup is a combination of a high intensity focused ultrasound (HIFU) system and a pulsed nanosecond laser system. The laser system has a 532 nm wavelength (Surelite SL III-10, Continuum, Santa Clara, CA, USA) with a pulse repetition frequency of 10 Hz and pulse duration of 3–5 ns. The laser system was triggered by a delay pulse generator (Model DG535, Stanford Research Systems, Sunnyvale, CA, USA). The same delay pulse generator was also used to trigger the HIFU system to temporally synchronize the ultrasound bursts and laser pulses. The trigger from the delay pulse generator was directed to a function generator (33250 A, Agilent Technologies, Santa Clara, CA, US) to produce a 0.5 MHz sine wave burst. The output burst from the function generator was first amplified by 50 dB through an RF amplifier (2100 L, Electronics & Innovation Ltd, Rochester, NY, US) and then passed to a HIFU transducer through an impedance matching circuit (Impedance Matching Network H107, Sonic Concepts, Bothell, WA, USA). The HIFU transducer (H-107, Sonic Concepts, Bothell, WA, US) has a center frequency of 0.5 MHz and radius of curvature of 63.2 mm with focal depth and focal width of 21.42 mm and 3.02 mm, respectively.

Figure 1.

Figure 1.

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Schematic of the experimental setup. Position 1 is for detecting photoacoustic signal and position 2 is for the HIFU treatment.

The sample and the HIFU transducer were submerged inside a water tank filled with degassed, deionized water such that the sample was at the focal spot of the transducer. The laser beam was focused by using a convex lens to result in a beam diameter of about 2 mm on the treatment area on the sample. The laser beam passed through the hole in the center of the HIFU transducer and was spatially aligned with the HIFU focal region on the sample before each treatment. For the spatial synchronization, the laser induced photoacoustic (PA) wave from the sample was detected by using the 0.5 MHz HIFU transducer. The detected PA wave was acquired by a digital oscilloscope (TBS 2000B, Tektronix Inc., Beaverton, OR, USA). The HIFU transducer was scanned across the sample cross-section until the maximum PA signal was detected, indicating that the focal region of the HIFU transducer overlaid the laser spot on the target. Apart from spatial synchronization, each laser pulse was also temporally synchronized with the ultrasound burst as described in (Qin et al 2020). Briefly, this was done by first precisely measuring the traveling time of the laser-induced PA wave propagating from the target to the HIFU transducer. Then this traveling time was given as a time delay to the delay generator, which triggered the laser beam. This time delay ensured that laser and ultrasound irradiated the sample at the same time. When detecting PA waves produced by 532 nm laser pulses, the produced signals were detected by the HIFU transducer and directed to the oscilloscope by connecting position 1 (figure 1). Position 2 connection was used to supply ultrasound sine wave burst to the HIFU transducer during the treatment.

For the treatment, ultrasound bursts of 1000 cycle were used at a burst repetition rate of 10 Hz (2% duty cycle) to minimize the effect of heating during the treatment. Before each treatment, the laser power was estimated at the sample surface by measuring the laser power at the output of the laser system and taking into account of the energy loss due to the glass tank wall in the laser propagation path. The laser energy loss due to the glass tank wall was determined by measuring the laser power before and after the glass tank wall.

The ultrasound peak negative pressure (PNP) was calibrated by using a needle hydrophone (HNR-0500, Onda, Sunnyvale, CA) before carrying out the experiment. During the calibration process, the needle hydrophone was positioned at the focus of the HIFU transducer inside a water tank filled with degassed, deionized water. Ultrasound bursts of 10 cycle were used during this process and the highest PNP calibrated was 5.4 MPa. No shock wave was detected, although the ultrasound waveform at the focus shows some distortions due to nonlinearity at high ultrasound pressure.

2.2. Treatment procedure and evaluation method

The treatment was carried out on two different types of samples: pork belly fat and human carotid artery plaque. First the treatment was carried out on pork belly fat samples to titrate the ultrasound and laser parameters for lipid removal. The first few pork belly fat samples were treated with high PNP ultrasound-only to find an ultrasound amplitude which does not result in any treatment effect on the sample. It was found that an ultrasound PNP of less than 2.94 MPa did not remove any lipid from the sample. The same experiment when repeated with laser-only treatment found laser pulse energy less than 60 mJ (19 mJ mm−2 with a focal spot of 2 mm in diameter) to be ineffective in producing any treatment effect on pork belly fat samples. For the next experiment on pork belly fat samples, the ultrasound PNP of less than 2.94 MPa and laser pulse energy of less than 60 mJ were used.

The experiment on pork belly fat samples was conducted in three different settings. In the first setting, the samples were treated with ultrasound-only, laser-only and combined ultrasound and laser using ultrasound amplitude of 2.45 MPa and laser pulse energy of 50 mJ (Results 3.1.1). The area of the fat tissue being removed from the samples was measured to compare the treatment effect between ultrasound-only, laser-only, and combined ultrasound and laser treatment. In the second setting, the samples were treated with a fixed laser pulse energy of 50 mJ but with different ultrasound PNPs of 0.49, 0.98, 1.47, 1.96 and 2.45 MPa (Results 3.1.2). In the third setting, the samples were treated with a fixed ultrasound PNP of 2.45 MPa but with different laser pulse energies of 10, 20, 30, 40 and 50 mJ (Results 3.1.3). The second and third settings were conducted to study the effects of change in ultrasound amplitude and laser pulse energy on the amount of lipid being removed from the pork belly fat sample. On each sample, the treatment was conducted on 2 different locations separated by at least 7.6 mm such that treatments do not affect each other, using 9000 ultrasound/laser pulses. Our previous study showed that the treatment effect would be significantly enhanced if the laser irradiated on the negative phase of a ultrasound cycle (Qin et al 2020). To ensure the laser temporal synchronization on the negative phase of ultrasound for at least half of the total treatment time, 4 different temporal synchronization were used for each treatment. For a 0.5 MHz ultrasound wave, the temporal synchronization was changed by 1/4 (0.5 μs out of 2 μs) of ultrasound wave time period after every 1/4 (2250 out of 9000 ultrasound/laser pulses) of treatment time during the combined ultrasound and laser treatment. In the first setting, 6 treatments were performed on 3 samples for combined ultrasound and laser treatment group, and 4 treatments on 2 samples for the ultrasound-only and laser-only groups. In the second and third settings, 4 treatments were performed on 2 samples for each treatment group. Minimum 4 treatments were carried out in each group to establish statistical significane between the treatment groups. After the treatment, the removal of fat created an elliptical shape cavity on the surface of the sample. The area of this cavity was used for quantification of the treatment results. This area of the cavity was marked on the pictures taken through a microscope. The major and minor diameter of the cavity were first measured by marking the boundary of the cavity in two directions perpendicular to each other, and then area of the ellipse (πab) was calculated. The depth of the elliptical cavity was not uniform due to the gaussian profile of the laser beam and ultrasound focal width, it is deeper in the center and less deep near the boundary.

After the pork belly fat sample experiment, the experiments were conducted on atherosclerotic plaque samples (Results 3.2). Human atherosclerotic plaque samples were collected at the University of Kansas Medical Center during carotid endarterectomy surgeries. All patient information was removed before samples were transferred out of the operation room. A total of 10 treatments were carried out on the samples, out of which 4 treatments were using combined ultrasound and laser, 3 using ultrasound-only and 3 using laser-only. A PNP of 5.4 MPa and laser pulse energy of 50 mJ were used for all the treatments. Only the lipid-rich regions of the samples were treated with 18 000 ultrasound/laser pulses during each treatment. To ensure the laser temporal synchronization on the negative phase of ultrasound, the temporal synchronization was shifted by 0.5 μs after every 4500 ultrasound/laser pulses during the combined ultrasound and laser treatment. To quantify the treatment outcomes, a circular section with 6 mm diameter or 28.27 mm2 area on the sample was selected as the targeted treatment zone, as shown in figure 6(f). This 6 mm diameter for treatment zone was selected based on the pork belly fat experiment, in which the treatment affected region was around 6 mm while using combined ultrasound and laser (figure 2(d)). The percentage of lipid-mass dissolved within this 6 mm circular treatment zone was used for comparing the treatment effects (figure 6(f)). The lipid-mass dissolved after treating the plaque sample with combined ultrasound and laser is marked with black boundary in figures 6(e) and (f).

Figure 6.

Figure 6.

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Carotid artery plaque samples before and after treatment with 18000 ultrasound/laser pulses using combined ultrasound and laser (a), (b); ultrasound-only (c); and laser-only (d). (e) Lipid removed during treatment of sample shown in (a) marked with black boundary. (f) Magnified view of (e) with treatment zone of 6 mm marked with green boundary and lipid removed within treatment zone marked with black boundary. (i) indicates before each treatment and (ii) indicates after each treatment. Ultrasound PNP: 5.4 MPa, Laser pulse energy: 50 mJ.

Figure 2.

Figure 2.

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Pork belly fat sample after treatment with 9000 ultrasound/laser pulses. (a) Ultrasound-only treatment, (b) laser-only treatment, (c) combined ultrasound and laser treatment, (d) magnified view of treated area in (c). Ultrasound PNP: 2.45 MPa, laser pulse energy: 50 mJ. Treatment area in (a), (b) and (c) are marked with red dash circle. Elliptical cavity used for quantification is marked with green ellipse. Major and minor diameter of ellipse are marked in blue lines.

During the evaluation of the treatment effects in the pork belly fat samples and the atherosclerotic plaque samples, in order to minimize the subjective bias in the quantification process, two readers who were blinded to the group information were used to verify the results.

3. Results

3.1. Pork belly fat sample

3.1.1. Treatment with ultrasound-only, laser-only and combined ultrasound and laser

Figure 2 shows the pork belly fat samples treated with ultrasound-only, laser-only, and combined ultrasound and laser. The ultrasound PNP of 2.45 MPa and laser pulse energy of 50 mJ (16 mJ mm−2) were used for all the treatments. No treatment effect was visible on samples treated with ultrasound-only (figure 2(a)) and laser-only (figure 2(b)), whereas, the samples treated with combined ultrasound and laser resulted in fat tissue removal from the samples (figures 2(c), (d)). The removal of fat tissue resulted in cavities on the sample surface. The area of surface cavity after the treatment was used to assess the treatment effect. Figure 3 shows the cavity area on samples after the treatment with ultrasound-only, laser-only and combined ultrasound and laser. Out of the 4 treatments carried out on samples using the laser-only, no cavity was observed in any case. For ultrasound-only, out of the 4 treatments, surface cavity of area 44 mm2 was observed in only one treatment. In contrast, for the 6 treatments with combined ultrasound and laser, cavities were observed in all cases with areas ranging from 105 to 147 mm2 and a mean cavity area of 124 mm2. The treatment area between the combined ultrasound and laser group and ultrasound/laser only group was statistically significant (p < 0.001).

Figure 3.

Figure 3.

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Pork belly fat area affected after 9000 ultrasound/laser pulses treatment with only ultrasound PNP of 2.45 MPa, only laser pulse energy of 50 mJ, and combined ultrasound PNP of 2.45 MPa and laser pulse energy of 50 mJ. US: ultrasound; ‘***’: p < 0.001.

3.1.2. Treatment with varying ultrasound PNP

Figure 4 shows the samples treated with combined ultrasound and laser at a fixed laser pulse energy but different ultrasound PNPs. The laser pulse energy was fixed at 50 mJ and ultrasound PNPs of 0.49, 0.98, 1.47, 1.96 and 2.45 MPa were used. The mean cavity area of fat tissue removal after treatment generally increased with the increase in ultrasound PNP. The mean cavity areas for 0.49, 0.98, 1.47, 1.96 and 2.45 MPa were 10, 0, 47, 72 and 124 mm2, respectively. The areas of fat tissue removal were statistically significant between ultrasound PNP 0.98 MPa and 1.47 MPa (p < 0.01), 1.47 MPa and 1.96 MPa (p < 0.05), and 1.96 MPa and 2.45 MPa (p < 0.001).

Figure 4.

Figure 4.

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Pork belly fat removal area after 9000 ultrasound/laser pulses treatment with combined ultrasound and laser. Laser pulse energy was kept at 50 mJ and different ultrasound PNP of 0.49, 0.98, 1.47, 1.96 and 2.45 MPa was used. ‘*’: p < 0.05; ‘**’: p < 0.01; ‘***’: p < 0.001.

3.1.3. Treatment with varying laser pulse energy

Figure 5 shows the samples treated with combined ultrasound and laser at a fixed ultrasound PNP but different laser pulse energy levels. The ultrasound PNP was fixed at 2.45 MPa and laser pulse energies of 10, 20, 30, 40 and 50 mJ were used. The mean cavity area of fat tissue removal after treatment generally increased with the increased laser pulse energy. The mean cavity areas of fat removal for 10, 20, 30, 40 and 50 mJ were 56, 70, 76, 70 and 124 mm2, respectively. Statistical significance was achieved for areas of fat tissue removal between 10 mJ and 20 mJ (p < 0.05), 40 mJ and 50 mJ (p < 0.001), 10 mJ and 30 mJ (p < 0.01), and 30 mJ and 50 mJ (p < 0.001).

Figure 5.

Figure 5.

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Pork belly fat removal area after 9000 ultrasound/laser pulses treatment with combined ultrasound and laser. Ultrasound PNP was fixed at 2.45 MPa and different laser pulse energies of 10, 20, 30, 40 and 50 mJ were used. ‘*’: p < 0.05; ‘**’: p < 0.01; ‘***’: p < 0.001.

3.2. Carotid artery plaque sample

Figure 6 shows the carotid artery plaque samples treated with combined ultrasound and laser, ultrasound-only and laser-only. The laser-only treatment was not able to remove any lipid-mass in plaque samples and less than 0.1 mm2 area was affected after the treatment. The ultrasound-only treatment removed small amounts of the lipid-mass from plaque samples, and the lipid-mass cross-section area of 2 to 5 mm2 was removed in 3 different treatments. In contrast, in the samples treated with combined ultrasound and laser, a lipid cross-section area of 12–20 mm2 was removed after the treatment.

Figure 7 shows the percentage of the lipid-mass dissolved in carotid artery plaque samples after treatment using combined ultrasound and laser, ultrasound-only and laser-only. After the treatment with laser-only, less than 0.4% of the lipid-mass in the treatment zone was removed. In case of treatment with ultrasound-only, 9% to 18% of lipid-mass was removed in the treatment zone. In contrast, in the samples treated with combined ultrasound and laser, 44% to 72% of the lipid-mass with a mean value of 58.38% was removed after the treatment. The percentage of lipid-mass removed between the combined ultrasound and laser and ultrasound/laser only group was statistically significant (p < 0.01). All the findings were verified by the two readers who were blinded to the treatment informaion. The readers also found significant more lipid-mass removal in the plaque samples treated with combined ultrasound and laser. Also, they did not found any lipid-mass removal in the samples treated with laser only and were only able to found slight lipid-mass removal in the samples treated with ultrasound when the treated area was marked.

Figure 7.

Figure 7.

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Percentage of lipid removed in carotid artery plaque samples after treatment with 18000 ultrasound/laser pulses using ultrasound-only, laser-only, and combined ultrasound and laser. Ultrasound PNP: 5.4 MPa, Laser pulse energy: 50 mJ, US: ultrasound.

4. Discussion

The results from the experiments on pork belly fat samples showed that the combined ultrasound and laser treatment was effective in disrupting fat tissues (figures 2, 3), whereas the same amplitude of ultrasound-only or same energy of laser-only was not able to disrupt the fat tissues. The treatment results between the control group and the combined ultrasound and laser group were statistically significant with p < 0.001 (figure 3). The results of the experiments using different ultrasound PNP and different laser pulse energies showed that fat removal was more affected by increasing ultrasound PNP as compared with laser power (figures 4, and 5). The removed fat area after treatment with different laser pulse energies was relatively unchanged for an increased laser pulse energy from 10 to 40 mJ. Based on these results, a higher amplitude of ultrasound (5.4 MPa) and the same laser energy (50 mJ) were used for experiments conducted on carotid artery plaque samples in the second part of the study. The results from the experiments on human carotid artery plaque samples showed that combined ultrasound and laser treatment removed a much larger area of the lipid core as compared with the same power of ultrasound-only and laser-only treatments (figures 6, 7).

The plaque-disrupting mechanism of the combined ultrasound and laser technology used in this study is similar to that in ELCA. ELCA uses high-power laser to disrupt the plaque through photochemical (Litvack et al 1988), photothermal (van Leeuwen et al 1993) and photomechanical (Topaz 1993) mechanisms. In ELCA, the laser thermal energy vaporizes the water creating a vapor bubble, which expands and collapses (photomechanically) to break the plaque (van Leeuwen et al 1993). In our technology, nanosecond pulsed laser irradiation nucleates the bubbles in the fluid through photothermal and photomechanic mechanisms (Hoffman and Telfair 2000). When a laser is superimposed on the rarefaction phase of an ultrasound wave, a high rarefaction pressure is generated due to combination of the ultrasound wave and laser-induced PA wave (Qin et al 2020). This high rarefaction pressure nucleates bubbles in the fluid, also known as the photospallation effect (Hoffman and Telfair 2000). In addition to photospallation, bubbles are formed as a result of vaporization of fluid due to laser-induced heating, which is similar to the photothermal mechanism in ELCA. Due to irradiation of ultrasound along with laser, these nucleated bubbles through photothermal and photospallation mechanisms expand and collapse in the ultrasound field and disrupt the plaque through mechanical forces (Wu and Nyborg 2008). In combined ultrasound and laser technology, the laser plays a critical role in initiation of cavitation through photothermal and photospallation mechanism, while the main role of ultrasound is to drive the formed cavitation and disrupt the plaque through mechanical forces. Cavitation is localized in the region where ultrasound and laser beams overlaid, and is mainly induced on the surface of the sample. The expansion and collapse of bubbles can disintegrate the surface of the sample into small pieces, and the surface will be eroded layer by layer. The speed of erosion will depend upon the treatment parameters, mainly on the pulse repetition rate. Also, stronger erosion may occur in the center of ultrasound and laser beams due to the presence of higher laser and ultrasound energy as a result of gaussian profile. Moreover, the use of ultrasound-only or laser-only do not generate strong enough cavitation to disrupt the sample surface.

The advantage of combining ultrasound with laser could be the reduced risk of complications as compared to the use of laser alone in ELCA. The usage of ELCA for atherosclerosis is low as compared to non-ELCA PCI due to the higher risk of complications such as dissection and perforation (Sintek et al 2021). The complications are partially due to strong vaporization and the uncontrolled forceful expansion of the vapor bubble and its collapse, resulting in shock waves (van Leeuwen Ton et al 1992). The chances of complications increase at high laser power and frequency rate. In our technology, low laser energy is needed to initiate the cavitation, which reduces the chances of complications. Also, bubble expansion and collapse can be better controlled by varying the ultrasound amplitude and number of cycles (Brujan et al 2005, 2008). A bubble initiated through low laser energy and controlled through driving ultrasound bursts has the potential to reduce the chances of vessel dissection and perforation during the treatment. In the experiments conducted on carotid artery plaque samples, the treatment effect was limited to the lipid-core, no damage to the fibrous tissue was observed (figure 6(a)). This technology is based on our previous developed USELT, which was tested to dissolve blood clots in veins (Jo et al 2021, Singh et al 2021). In our in vivo study using USELT, no damage to the vessel wall was observed in the vein section stained with H&E (hematoxylin and eosin) stain (Singh et al 2021). Based on our in vivo USELT study results, we expect less complications from the use of combined ultrasound and laser for the removal of atherosclerotic plaque. However, the exact possibility of vessel dissection and perforation during the atherosclerosis treatment can only be confirmed in future in vivo atherosclerosis studies.

The pulsed nanosecond laser technique used in this study was inspired by the ELCA laser system. However, the laser parameters were different than the laser system used in ELCA. The CVX-300 (Philips, Amsterdam, Netherlands) is the only laser system approved by the US Food and Drug Administration for ELCA. CVX-300 is an excimer laser, which emits 308 nm laser pulses of 125–200 ns pulse-width with a pulse repetition frequency of 25–80 Hz. The laser system (Surelite SL III-10, Continuum, Santa Clara, CA, USA) used in this study, emits a 532 nm laser pulse of 3–5 ns pulse-width with pulse repetition frequency of 10 Hz. A 532 nm wavelength was used due to its higher penetration depth than 308 nm (Oraevsky et al 1993). The high absorption of 308 nm by the lipid-core, restricts its penetration depth to less than 50 μm. The deeper penetration of 532 nm may result in less drastic and more uniform cavitation initiation. Uniform and controlled cavitation will disrupt the lipid-core with high efficiency and will reduce the chance of complications on the vessel as a result of bubble mechanical forces. A short laser pulse width of 3–5 ns was used in our study as it reduced the laser energy needed for initiating cavitation. The ELCA laser initiates cavitation by vaporizing water through a photothermal mechanism (van Leeuwen et al 1993). Whereas, for a short pulse-width nanosecond pulsed laser, cavitation can be initiated through a photospallation mechanism at a lower laser energy than required for photothermal mechanism (Hoffman and Telfair 2000). The CVX-300 delivers a laser fluence of 30–80 mJ mm−2 for ELCA whereas the laser fluence was not more than 30 mJ mm−2 in this study.

For clinical use, the laser in our technology can be directly delivered to the treatment site in the vessel using the endovascular laser catheter, like the current clinical treatment techniques such as ELCA (Cook et al 1991) and laser thrombolysis (den Heijer et al 1994). Apart from laser, the delivery of ultrasound during treatment in our technique will be less complex and it can be applied non-invasively from outside the body. Due to its similarity to the current clinical techniques such as catherter-based laser ablation and non-invasive application of ultrasound, our technology has high clinical potential. Also, our technology does not require any external addition of microbubble/contrast agents to generate cavitation. Some ultrasound-based technique require external injection of microbubbles to the target site to produce enhanced cavitation at low ultrasound energy (Molina et al 2009, Damianou et al 2015). The external injection of microbubble restricts the treatment time window as treatment can only be carried out when bubbles are present at the target site. Moreover, the treatment is severely affected by the density of bubbles present at the treatment site, which is affected by the blood flow as bubbles are continuously carried away by the blood. In our technology, bubbles can be continuously generated at the target location using the combination of nano-second laser pulses and ultrasound. Also, there is no limit to the number of bubbles produced, ensuring enough bubbles for disrupting blood clots and atherosclerotic plaques.

One limitation of this study is the use of water instead of blood for the experiment, which may resulting in overestimating the cavitation effect. The large uniaxial extensional viscosity of blood causes cavitation attenuation in blood (Brujan 2019). However, for bubbles larger than 50 μm in size, the effect of blood rheology is not significant (Kim 1994). Our previous studies have shown bubble size can increase up to 100 times on simultaneous irradiation of ultrasound and laser (Li et al 2018, Singh et al 2020). Moreover, the blood has much higher laser absorption than water, which can compensate for reduced cavitation effects due to blood rheology, even at much lower laser energy than used here.

The second limitation of this study is the assessment of the treatments results. We evaluated the treatment result by marking the affected area in the pictures taken before and after the treatment. Other quantification approach such as sample weighting were also tried for the quantification of treatment outcome during the study. However, the weight of the plaque sample was less than 0.5 gram and was very sensitive to the water content in the sample. The sample weight measurement was unreliable for treatment assessment as it changed drastically with slight change in water content in a sample. As a result, we only evaluated the area removed after treatment during the quantification process, and outcomes were also verified by two readers who were blinded to the treatment group information. This evaluation method, however, could underestimate the treatment effect because the depth of the treated area was not considered. A high-resolution 3D volumetric imaging technique could be a better approach for evaluation in the future.

5. Conclusion

This is the first study to test the combined ultrasound and laser-based technique for the dissolving of the atherosclerotic plaque. The results show that combined ultrasound and laser were more effective in removing atherosclerotic plaque’s lipid core as compared to the use of same energy of laser alone and same amplitude of ultrasound alone. The addition of ultrasound to laser reduced the laser energy required for the removal of atherosclerotic plaque, which can have some advantages over the current laser-only based clinical technique. The complications associated with current high-power laser-only techniques may be reduced and better plaque removal efficiency may be achieve. In the future, in vivo study should be conducted to further test the combined ultrasound and laser technique and verify its advantages in removing atherosclerotic plaques.

Data availability statement

The data cannot be made publicly available upon publication because they are not available in a format that is sufficiently accessible or reusable by other researchers. The data that support the findings of this study are available upon reasonable request from the authors.

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Associated Data

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Data Availability Statement

The data cannot be made publicly available upon publication because they are not available in a format that is sufficiently accessible or reusable by other researchers. The data that support the findings of this study are available upon reasonable request from the authors.

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