Sonodynamic Therapy: Advances and Challenges in Clinical Translation.

Sonodynamic therapy (SDT) consists of the synergetic interaction between ultrasound and a chemical agent. In SDT, the cytotoxicity is triggered by ultrasonic stimuli, notably through cavitation. The unique features of SDT are relevant in the clinical context more than ever: the need for efficacy, accuracy, and safety while being noninvasive and preserving the patient's quality of life. However, despite the promising results of this technique, only a few clinical reports describe the use of SDT. The objective of this article is to provide an extensive overview of the clinical and preclinical research conducted in vivo on SDT, to identify the limitations, and to detail the developed strategies to overcome them.

Focused ultrasound transducer for thermal treatment.

Air-backed transducers have been employed for thermal ultrasonic treatment including both ablation and hyperthermia because the power efficiency rather than the bandwidth is a main concern, unlike a typical imaging transducer working in a pulse mode. The characteristic of an air-backed piezoelectric transducer with a matching layer is analysed, and the role and choice of the matching layer is discussed. An element size of a focused array transducer, appropriate for such thermal treatment, is then estimated, and the characteristic of a piezoceramic transducer element of such a size was numerically analysed using a finite element code. The characteristic of a piezocomposite transducer element is also numerically analysed and its suitability to such a therapeutic array transducer is discussed.

High-speed observation of bubble cloud generation near a rigid wall by second-harmonic superimposed ultrasound.

Cavitation bubbles are known to accelerate therapeutic effects of ultrasound. Although negative acoustic pressure is the principle factor of cavitation, positive acoustic pressure has a role for bubble cloud formation at a high intensity of focused ultrasound when cavitation bubbles provide pressure release surfaces converting the pressure from highly positive to negative. In this study, the second-harmonic was superimposed onto the fundamental acoustic pressure to emphasize either peak positive or negative pressure. The peak negative and positive pressure emphasized waves were focused on a surface of an aluminum block. Cavitation bubbles induced near the block were observed with a high-speed camera by backlight and the size of the cavitation generation region was measured from the high-speed images. The negative pressure emphasized waves showed an advantage in cavitation inception over the positive pressure emphasized waves. In the sequence of the negative pressure emphasized waves immediately followed by the positive pressure emphasized waves, cavitation bubbles were generated on the block by the former waves and the cavitation region were expanded toward the transducer in the latter waves with high reproducibility. The sequence demonstrated its potential usefulness in enhancing the effects of therapeutic ultrasound at a high acoustic intensity.

Changes in backscatter of liver tissue due to thermal coagulation induced by focused ultrasound.

Ultrasonic imaging has advantages in its self-consistency in guiding and monitoring ultrasonic treatment such as high-intensity focused ultrasound (HIFU) treatment. Changes in ultrasonic backscatter of tissues due to HIFU treatment have been observed, but their mechanism is still under discussion. In this paper, ultrasonic backscatter of excised and degassed porcine liver tissue was observed before and after HIFU exposure using a diagnostic scanner, and its acoustic impedance was mapped using an ultrasonic microscope. The histology of its pathological specimen was also observed using an optical microscope. The observed decrease in backscatter intensity due to HIFU exposure was consistent with a spatial Fourier analysis of the histology, which also showed changes due to the exposure. The observed increase in acoustic impedance due to the exposure was also consistent with the histological change assuming that the increase was primarily caused by the increase in the concentration of hepatic cells.

Investigation of feasibility of noise suppression method for cavitation-enhanced high-intensity focused ultrasound treatment.

In high-intensity focused ultrasound (HIFU) treatment, a method that monitors tissue changes while irradiating therapeutic ultrasound is needed to detect changes in the order of milliseconds due to thermal coagulation and the presence of cavitation bubbles. The new filtering method in which only the HIFU noise was reduced while the tissue signals remained intact was proposed in the conventional HIFU exposure in our preliminary study. However, HIFU was irradiated perpendicular to the direction of the imaging ultrasound in the preliminary experiment, which was believed to be impractical. This study investigated the efficacy of the proposed method a parallel setup, in which both HIFU and imaging beams have the same axis just as in a practical application. In addition, this filtering algorithm was applied to the "Trigger HIFU" sequence in which ultrasound-induced cavitation bubbles were generated in the HIFU focal region to enhance heating. In this setup and sequence, HIFU noise level was increased and the summation or difference tone induced by the interaction of HIFU waves with the imaging pulse has the potential to affect this proposed method. Ex-vivo experiments proved that the HIFU noise was selectively eliminated by the proposed filtering method in which chaotic acoustic signals were emitted by the cavitation bubbles at the HIFU focus. These results suggest that the proposed method was practically efficient for monitoring tissue changes in HIFU-induced cavitation bubbles.

Ultrasound Sub-pixel Motion-tracking Method with Out-of-plane Motion Detection for Precise Vascular Imaging.

Ultrasound vascularity imaging provides important information for differential diagnosis of tumors. Peak-hold (PH) is a useful technique for precisely imaging small vessels by selecting a maximum brightness in each pixel through the frames obtained sequentially. To use PH successfully one needs motion compensation to reduce image blur, but out-of-plane motion cannot be avoided. To address this problem, we developed a sub-pixel motion-tracking method with out-of-plane motion detection (OPMD). It is a combination of the sum of the absolute differences (SAD) method and the Kanade-Lucas-Tomasi method and can be accurately applied to various motions. The value from OPMD (γ) is defined as a statistical value obtained from the distribution of residual values in the SAD procedure with the obtained frames. The value is ideally 0, and the frames having large γ are removed from the PH procedure. The accuracy of the proposed tracking method was found by a simulation study to be approximately 20 µm. We also found, through a phantom experiment, that the value of γ sensitively increased enough to detect out-of-plane motion. Most important, γ begins to increase before tracking errors occur. This suggests that OPMD can be used to predict tracking errors and effectively remove frames from the PH procedure. An in vivo experiment with a rabbit showed that the PH image obtained with motion tracking clearly revealed peripheral vessels that were blurred in the PH image obtained without motion tracking. We also found that the image quality becomes better when OPMD was used to remove frames including out-of-plane motion.

Sonodynamically-induced Anticancer Effects of Polyethylene Glycol-Modified Carbon Nano Tubes.

BACKGROUND/AIM: Sonodynamic cancer therapy is based on the preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and the subsequent activation of the drug by ultrasound irradiation. In the present study, we investigated the sonodynamically-induced antitumoral effect with functionalized carbon nanotubes, such as poly-ethylene glycol-modified carbon nanotubes (PEG-modified CNTs). MATERIALS AND METHODS: Antitumor effects were evaluated using histological observation and assessing tumor growth following sonodynamic exposure to PEG-modified CNTs. RESULTS: The combined treatment of 100 µM PEG-modified CNT and ultrasound induced a 2-fold cytotoxicity. Sodium azide, which quenches singlet oxygen, significantly inhibited ultrasonication induced cell damage in the presence of PEG-modified CNTs. This suggests that singlet oxygen produced by the combined use of PEG-modified CNTs and ultrasound is involved in the induction of antitumoral effects. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with PEG-modified CNTs, while neither the treatment with PEG-modified CNTs alone nor ultrasound alone caused any necrosis. CONCLUSION: These results indicate that PEG-modified CNT functions as a sonosensitizer and is effective for sonochemical treatment of solid tumors.

Sonodynamic therapy consisting of focused ultrasound and a photosensitizer causes a selective antitumor effect in a rat intracranial glioma model.

UNLABELLED: In this study we sought to determine the optimal focused ultrasound acoustic conditions with photosensitizers for the ablation of experimental intracranial glioma in rats. MATERIALS AND METHODS: Normal rat brains were sonicated via a transducer placed on the dural surface with or without a prior intravenous injection of the photosensitizer Rose Bengal (50 mg/kg of body weight). The ultrasound intensity was varied to 25, 110 or 150 W/cm(2), and the duration of irradiation was 10 s, or 1, 3, or 5 min. In experimental intracranial gliomas, one week after inoculation of C6 rat glioma cells in the rat brain, the rat brain was sonicated through a 10 mm-diameter craniotomy. RESULTS: A selective antitumor effect against cerebral glioma while sparing normal brain tissues was achieved by sonodynamic focused therapy consisting of focused ultrasound at 25 W/cm(2) at 1 MHz for 5 min and Rose Bengal (50 mg/kg of body weight). The areas of tumors in sham-operated rats and in rats that received sonodynamic therapy without and with Rose Bengal at an intensity of 25 W/cm(2) for 5 min were 19.53+/-3.89, 10.64+/-2.21 and 3.01+/-1.74 mm(2), respectively. The tumor area was significantly smaller in the ultrasound therapy groups than in control non-treated animals (p=0.002). There was no significant temperature change in tumor tissues during sonication with 25 W/cm(2) at 1 MHz. CONCLUSION: This is the first report to demonstrate the usefulness of sonodynamic therapy consisting of focused ultrasound and photosensitizer for the treatment of experimental malignant glioma.

Effects of focused ultrasound sonodynamic treatment on the rat blood-brain barrier.

BACKGROUND: Ultrasound has recently been applied to the treatment as well as the diagnosis of various pathologies, and its antitumor effects in the treatment of human cancer and experimental models of cancer have been demonstrated. In addition, it is possible that certain photosensitizers will enhance the antitumor effects of ultrasound. However, very few studies have been reported on how the blood-brain barrier is affected by sonodynamic therapy. The purpose of this study was to evaluate disruption of the blood-brain barrier with focused ultrasound with a photosensitizer, for clinical application of sonodynamic therapy to brain tumors. MATERIALS AND METHODS: Rat brains were subjected to focused ultrasound irradiation via a transducer with or without prior intravenous injection of photosensitizer, and lesions were examined histologically by electron microscopy. RESULTS: Electron microscopically, swelling of astroglial processes, denatured cells, protoplasm of endothelial cells, and mitochondria were observed in the center and border of regions of ultrasonic irradiation. There were numerous pinocytotic vesicles in the cytoplasm of the endothelial cells. In addition, disruption of the cytoplasmic membrane of endothelial cells and astroglia was found in these regions. CONCLUSION: These findings suggest that sonodynamic therapy with a photosensitizer affects the blood-brain barrier, and that blood vessel permeability increases not only as a result of destruction of the blood-brain barrier but also by disruption of the cytoplasmic membrane of endothelial cells.

Sonodynamically-induced apoptosis, necrosis, and active oxygen generation by mono-l-aspartyl chlorin e6.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of a photochemically active chlorin, mono-l-aspartyl chlorin e6 (NPe6). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of NPe6, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 80 microM NPe6 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound alone, at the same intensity, or NPe6 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and NPe6 but not in cells treated with ultrasound or NPe6 alone. In addition, NPe6 substantially enhanced nitroxide generation by ultrasound in the same acoustical arrangement. Sonodynamically-induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results suggest that the combination of ultrasound and NPe6 sonochemically induces apoptosis as well as necrosis in HL-60 cells. They further suggest that some ultrasonically-generated active species, deactivatable by histidine, are the major mediators to induce the observed apoptosis.

Sequential changes in rat femoral artery blood flow and tissue degeneration after exposure to high-intensity focused ultrasound.

PURPOSE: The purpose of this study was to investigate the sequential changes in rat artery blood flow and tissue degeneration after exposure to high-intensity focused ultrasound (HIFU) in vivo. METHODS: HIFU was applied through the skin to the femoral artery of Sprague-Dawley rats. The peak intensities used were 530, 1080, 2750, and 4300 W/cm(2). After exposure, we measured the peak systolic velocity (PSV) in the artery every 1 min until the velocity stabilized. The vessel was resected and examined histologically 7 days after exposure. RESULTS: PSV was not significantly affected by HIFU exposure at 530 W/cm(2). PSV increased immediately after HIFU exposure at intensities of 1080 and 2750 W/cm(2). PSV after HIFU exposure at 1080 W/cm(2) fell to the control level within minutes; however, PSV increased immediately after HIFU exposure at 2750 W/cm(2) and then decreased slowly but remained at a higher level than the control for 15 min. On HIFU exposure at 4300 W/cm(2), the target artery was completely occluded. Histological studies 7 days after HIFU exposure demonstrated that exposure at 530 and 1080 W/cm(2) induced vacuolar degeneration in the tunica media of the femoral artery in rats; exposure to HIFU at 2750 and 4300 W/cm(2) resulted in strong necrotic degeneration in the tunica media. These histological changes were more marked than those found immediately after HIFU exposure. Organized thrombus formation was observed only for HIFU exposure at 4300 W/cm(2). CONCLUSION: Sequential changes in arterial blood flow after HIFU exposure vary with the intensity, and the histological changes in arterial tissue progress over time. These phenomena should be considered when HIFU is clinically applied to achieve arterial occlusion.

Cavitation-threshold Determination and Rheological-parameters Estimation of Albumin-stabilized Nanobubbles.

Nanobubbles (NBs) are of high interest for ultrasound (US) imaging as contrast agents and therapy as cavitation nuclei. Because of their instability (Laplace pressure bubble catastrophe) and low sensitivity to US, reducing the size of commonly used microbubbles to submicron-size is not trivial. We introduce stabilized NBs in the 100-250-nm size range, manufactured by agitating human serum albumin and perfluoro-propane. These NBs were exposed to 3.34- and 5.39-MHz US, and their sensitivity to US was proven by detecting inertial cavitation. The cavitation-threshold information was used to run a numerical parametric study based on a modified Rayleigh-Plesset equation (with a Newtonian rheology model). The determined values of surface tension ranged from 0 N/m to 0.06 N/m. The corresponding values of dilatational viscosity ranged from 5.10-10 Ns/m to 1.10-9 Ns/m. These parameters were reported to be 0.6 N/m and 1.10-8 Ns/m for the reference microbubble contrast agent. This result suggests the possibility of using albumin as a stabilizer for the nanobubbles that could be maintained in circulation and presenting satisfying US sensitivity, even in the 3-5-MHz range.

Visualization of murine lymph vessels using photoacoustic imaging with contrast agents.

Metastasis frequently occurs even in the early stage of breast cancer. This research studied the feasibility of using photoacoustic (PA) imaging for identifying metastasis in the lymph vessels of mice. The photoacoustic efficiency of various contrast agents was investigated, and the influence of scattered light was evaluated by using a lymph vessel phantom. The lymph vessels of mice were then visualized using the selected contrast agents: indocyanine green (ICG) and gold nanorods (AuNR). The attenuation of the PA imaging was -1.90 dB/mm, whereas that of the fluorescence imaging was -4.45 dB/mm. The results indicate the potential of identifying sentinel lymph nodes by using PA imaging with these contrast agents.

Evaluation of a Three-Hydrophone Method for 2-D Cavitation Localization.

Cavitation is a critical parameter in various therapeutic applications involving ultrasound (US) such as histotripsy, lithotripsy, drug delivery, and cavitation-enhanced hyperthermia. A cavitation exposure outside the region of interest may lead to suboptimal treatment efficacy or in a worse case, to safety issues. Current methods of localizing cavitation are based on imaging approaches, such as beamforming the cavitation signals received passively by a US imager. These methods, although efficient, require expensive equipment, which may discourage potential future developments. We propose a three-hydrophone method to localize the cavitation cloud source. First, the delays between the three receptors are measured by detecting the maximum of their intercorrelations. Then, the position of the source is calculated by either minimizing a cost function or solving hyperbolic equations. After a numerical validation, the method was assessed experimentally. This method was able to track a source displacement with accuracy similar to the size of the cavitation cloud (2-4 mm). This light and versatile method provides interesting perspectives since localization can be executed in real time, and the extension to 3-D localization seems straightforward.

Evaluation of a Three Hydrophones Method for 2-Dimensional Cavitation Localization.

Cavitation is a critical parameter in various therapeutic applications involving ultrasound (US) such as histotrispy, lithothripsy, drug delivery, and cavitation-enhanced hyperthermia. A cavitation exposure outside the region of interest may lead to suboptimal treatment efficacy or in a worse case, to safety issues. Current methods of localizing cavitation are based on imaging approaches, such as beamforming the cavitation signals received passively by a US imager. These methods, although efficient, require expensive equipment, which may discourage potential future developments. We propose a threehydrophone method to localize the cavitation cloud source. Firstly, the delays between the three receptors are measured by detecting the maximum of their inter-correlations. Then, the position of the source is calculated by either minimizing a cost function or solving hyperbolic equations. After a numerical validation, the method was assessed experimentally. This method was able to track a source displacement with accuracy similar to the size of the cavitation cloud (2-4 millimeters). This light and versatile method provides interesting perspectives since localization can be executed in real time and the extension to three-dimensional localization seems straightforward.

Sonodynamic therapy on chemically induced mammary tumor: pharmacokinetics, tissue distribution and sonodynamically induced antitumor effect of gallium-porphyrin complex ATX-70.

Sonodynamically induced antitumor effect of a gallium porphyrin complex, ATX-70 was evaluated on a chemically induced mammary tumor in Sprague-Dawley rats. The timing of 24 h after the administration of ATX-70 was chosen for ultrasonic exposure, based on pharmacokinetic analysis of ATX-70 concentrations in the tumor, plasma, skin, and muscle. At an ATX-70 dose not less than 2.5 mg/kg and at a free-field ultrasonic intensity not less than 3 W/cm(2), the synergistic effect between ATX-70 administration and ultrasonic exposure on the tumor growth inhibition was significant. These results suggest that ATX-70 is a potential sonosensitizer for sonodynamic treatment of spontaneous mammary tumors.

Blood flow occlusion via ultrasound image-guided high-intensity focused ultrasound and its effect on tissue perfusion.

This study investigated the induction of tissue necrosis by arterial blood flow occlusion using ultrasound image-guided high-intensity focused ultrasound (HIFU). We constructed a prototype HIFU transducer in combination with an imaging probe that provided color Doppler imaging and ultrasound contrast imaging. The HIFU beam was aimed into a branch of the renal artery in vivo. The renal artery branches of eight rabbits were occluded by HIFU at an intensity of 4 kW/cm(2) (from 2 to 10 times of each sonication for 5 s). When the HIFU exposure was successful, complete cessation of blood flow was observed by color Doppler imaging with success rate of 100% (8/8). Furthermore, lack of perfusion was observed in the renal cortex with a contrast-enhanced image. Postmortem histologic evaluation showed a wedge-shaped area of infarction in six of seven cases, corresponding to the lack of the contrast medium in the ultrasound image. These results demonstrated that ultrasound image-guided HIFU can be used to induce arterial occlusion, thus producing infarction and necrosis of the perfused tissue.

Development of Real-Time 3-D Photoacoustic Imaging System Employing Spherically Curved Array Transducer.

Photoacoustic (PA) imaging is a promising imaging modality to visualize specific living tissues based on the light absorption coefficients without dyeing. In this paper, a real-time PA imaging system with a tunable laser was newly developed with an originally designed spherically curved array transducer. Five different series of experiments were conducted to validate the PA measurement system. The peak frequency of the transducer response was 17.7 MHz, and a volume-imaging rate of 3-D volume imaging was 10-20 volumes per second. The spatial resolution of imaging was 90- [Formula: see text] along both the axial and lateral directions. The developed imaging system could measure a difference on an absorption coefficient of gold nanorods. Additionally, the PA imaging could visualize the in vivo microvasculatures of a human hand. This PA imaging system with higher spatial-temporal resolution and the tunable laser further should enhance our understanding of not only basic properties of the photo acoustics but also clinical applications.

An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells.

To verify the effect of echo-contrast agent (ECA) on apoptosis induced by ultrasound, leukemia cell lines (Jurkat, Molt-4 and U937) were sonicated at intensities previously shown to induce optimal apoptosis with or without Levovist, an ECA. The results showed that loss of viability and apoptosis can be induced in all three cell lines, apoptosis highest with Molt-4, based on viability and DNA fragmentation assay. Such finding was supported by corresponding increase of cells with low mitochondrial membrane potential, high superoxide production, increased intracellular calcium concentration, and phosphorylation of histone H2AX after sonication. Optimal ultrasound condition was 0.3W/cm(2), 1MHz, 10% duty factor pulsed at 100Hz; but in the presence of Levovist, an apparent shift of cell killing induction was observed at 0.2W/cm(2). While these results further confirmed previous findings on ultrasound-induced apoptosis, they also suggest that use of an enhancing factor, such as addition of ECA, may be useful in cancer therapy when a much lower intensity is desired.