Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound
Introduction
In recent years, ultrasound therapy for the treatment of tumors has been successfully developed. For example, high-intensity focused ultrasound (HIFU) has been used to treat solid tumors and its efficacy and safety have been confirmed by clinical trials [1]. On the other hand, experiments related to the biological effects of relatively low-level ultrasound on malignant tissues and their clinical applications are still being investigated [2], [3]. One of the most attractive methods in low-intensity ultrasound therapy is sonodynamic therapy (SDT). In SDT, therapeutic effects can be mediated by free radicals, especially hydroxyl radicals and singlet oxygen [2], [3], [4]. Other species such as hydrogen peroxide, singlet oxygen, and superoxide ions are formed later under certain conditions [2], [3], [4], [5], [6]. Other chemicals known as sonosensitizers can be chemically activated by exposure to ultrasound. Sonodynamic therapy results from the non-thermal effects of ultrasound, especially cavitation [2], [3], [6], [7], [8], [9], [10], [11].
Historically, cavitation bubbles have been classified into two types, inertial and non-inertial. Inertial cavitation bubbles are defined as bubbles that are nucleated in solution and undergo violent collapse to produce localized high-temperature and pressure. The temperature and pressure experienced by the material contained within the imploding cavities may reach values in excess of 5000 K and 800 atm [12], [13], [14]. These extreme conditions may induce a multitude of chemical reactions within and surrounding the bubbles, including a concentration of energy sufficient to generate light, or sonoluminescence. Acoustic inertial cavitation generates free radicals from the breakdown of water and other molecules. The initial step in the decomposition of water is the production of hydroxyl and hydrogen radicals [2], [3], [4], [6], [7], [8], [9], [10], [11]. Many tests for the detection of cavitation are possible based on measurements of free radical concentration and chemical reaction products [15], [16], [17]. In order to quantify the effects of exposure parameters under therapeutic conditions such as sonodynamic therapy, it is necessary initially to evaluate the inertial cavitation activity in vitro. It has been shown that terephthalic acid is suitable for ionizing radiation dosimetry and also for detecting and quantifying free hydroxyl radicals generated by the collapse of cavitation bubbles in ultrasound fields [15], [16], [17], [18].
A number of investigators have shown that the acoustic cavitation can more easily be induced by standing waves than by progressive waves [14], [19], [20]. However, irradiation with standing waves does not seem to be widely applicable to various therapeutic settings because tumor sites are quite varied, thus limiting the clinical applications. Therefore, in this study we opted to use the progressive wave mode.
The experimental results from studies in sonochemistry indicate that activity of cavitation generated by multi-frequency ultrasound irradiation is higher than by single-frequency irradiation [21], [22], [23], [24]. Feng et al. [21] have shown based on Gaussian distribution of cavitation bubble radii, that cavitation bubbles with the resonance frequency of about 159 kHz are present in the cavitation zone of normal water solution with the highest probability. On the other hand, Matsumoto et al. [25] showed that the acoustic cavitation induced by ultrasound is considered to be strongly dependent on ultrasound frequency. Also, the frequency response of bubbles has dependence on the ambient ultrasound pressure. At 50–100 kPa of ambient amplitude, the range of frequencies that reach the collapsing phenomenon of cavitation bubbles in ultrasound field are at 50–200 kHz in frequency. Therefore, if the frequency is selected carefully, it is technically possible to concentrate energy in spatially restricted area in a media such as water or human tissue. Then, we have selected 150 kHz. However, given the capability of low-frequency ultrasound to produce cavitation, we believe that by combining 150 kHz ultrasound with a low-MHz frequency, even the low-level intensity may be of use in SDT.
In this study, the cavitation activity in dual-frequency fields (150 kHz and 1 MHz) was the subject of investigation to determine the influence of the exposure parameters in several combinations, mode of sonication, duty cycle, intensity and duration at low-level intensities (I ⩽ 3 W/cm2).
Section snippets
Dosimetry solution
A solution of terephthalic acid (TA) was prepared according to the standard protocol. Terephthalic acid, (Aldrich, 0.3323 g, 2 × 10−3 mol), was dissolved by heating and 0.200 g (5 × 10−3 mol) NaOH and phosphate buffer (pH = 7.4) (prepared from KH2PO4 0.58 g (4.4 × 10−3 mol) and Na2HPO4 (0.98 g, 7.6 × 10−3 mol)) were added. The resulting solution was then diluted to 1 dm3 with distilled and deionized water. Before use, the solution was kept in a refrigerator (∼4 °C) and in the dark to prevent photochemical
1 MHz ultrasound sonication
The results of experiments related to sonication by 1 MHz ultrasound field (ISATA = 2 W/cm2 and sonication duration 20 min) are shown in Table 1. As can be seen from the Table the amount of fluorescence intensity generated by 1 MHz continuous ultrasound field is significantly higher than by the pulse mode at different duty cycles (p-value < 0.05).
In Table 2, we have compensated sonication duration by 1 MHz pulsed irradiation at different duty cycles (ISATA = 2 W/cm2).
It can be seen that with compensation
Discussion
In summary, the above-mentioned experiments show that the combined low-level irradiation of dual-frequency ultrasound remarkably enhances the fluorescence intensity that, in turn, demonstrates the enhancement of the inertial cavitation activity under the conditions used in this study. With equal energy, the combination mode of sonication caused a fluorescence intensity of about 1.5 times higher than that obtained with the algebraic sum of the 150 kHz and 1 MHz irradiations, and also 3.5 times the
Conclusion
In the current work, we have demonstrated that simultaneous combined sonication with 1 MHz and 150 kHz irradiation induces inertial cavitation, even at low-level intensities (I < 3 W/cm2). At various duty cycles for each probe, the fluorescence intensity increased with higher duty cycles (80% > 50% > 20% at 1 MHz and 50% > 33% > 25% at 150 kHz). Also, in the continuous mode of sonication the fluorescence intensity is higher than in the pulsed mode. The inertial cavitation activity at higher ultrasound
Acknowledgements
The authors greatly appreciated the help of Professor Timothy J. Mason at Coventry University for the preparation and synthesis of the standard solution of 2-hydroxyterephthalic acid.
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