Ultrasonics Sonochemistry xxx (2015) xxx–xxx

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The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals Haobo Xu a,b,1, Xin Sun a,b,1, Jianting Yao a,1, Jian Zhang b, Yun Zhang a, Haibo Chen a, Juhua Dan b, Zhen Tian b, Ye Tian a,b,⇑ a b

Department of Cardiology, The First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin 150001, China Key Laboratory of Cardiovascular Pathophysiology, State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Harbin Medical University, Harbin 150081, China

a r t i c l e

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Article history: Received 30 July 2014 Received in revised form 11 April 2015 Accepted 18 April 2015 Available online xxxx Keywords: Ultrasound Protoporphyrin IX Hydroxyl radical Inertial cavitation Sonochemistry

a b s t r a c t The ultrasound activation of certain drugs, such as porphyrins, could cause synergistic cytotoxic effects on cells. Both sonomechanical and sonochemical effects occur and the latter play a critical role because antioxidant agents could exert significant protective effects against the cytotoxicity. To investigate the reactive oxygen species involved in the sonochemical effects, aqueous protoporphyrin IX (PpIX) solutions were characterized under ultrasound sonication in this study. Inertial cavitation was indirectly evaluated using terephthalic acid dosimetry. The fluorescence intensity of the PpIX was measured using a fluorescence spectrophotometer. The effects of PpIX concentration, ultrasound parameters and free radical scavengers on the PpIX activation by ultrasound were investigated. Our results showed that the increase in PpIX decomposition was significantly correlated with cavitation activities (R = 0.9874, p < 0.05), and the decomposing effect increases with ultrasound intensity (0.6–1.5 W/cm2), initial PpIX concentration (1–5 lM), duty cycle (10–100%) and the sonication duration (2–10 min). The fluorescence and absorption spectra of PpIX showed a decrease in the peak intensity without spectral shifts or new peak build-up after sonication. The PpIX decomposition was significantly inhibited by hydroxyl radical scavengers, histidine, mannitol, acetone, methanol and ethanol, but the decomposition was not inhibited by sodium azide, catalase or superoxide dismutase. These results suggest that the decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, which sheds some light on the sonochemical effects of the interaction between ultrasound and porphyrins. Ó 2015 Published by Elsevier B.V.

1. Introduction Ultrasound has been used in medicine for diagnostic imaging applications, facilitating the delivery of drugs, promoting wound healing as well as directly tumor ablations [1,2]. Ultrasound induces cell-damage through mechanical forces (i.e., microstreaming, shear stress and bubble-cell collisions) and chemical toxins (i.e., free radicals and peroxides) [3,4]. The mechanical and chemical damages caused by ultrasound depend strongly on the ultrasound frequency and intensity, because various types of cavitation bubbles occur under different sonication conditions that determine the types of damage [3]. Two types of cavitation bubbles have been categorized to aid in understanding the biological effects ⇑ Corresponding author at: Department of Cardiology, The First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, 23 Youzheng Street, Harbin 150001, China. Tel.: +86 451 85555943; fax: +86 451 87530341. E-mail address: [email protected] (Y. Tian). 1 Haobo Xu, Xin Sun and Jianting Yao contributed equally to this study.

of ultrasound, non-inertial and inertial [4]. Non-inertial cavitation bubbles are defined as microbubbles that oscillate around an equilibrium radius without undergoing violent collapse. Inertial cavitation bubbles are defined as bubbles that are nucleated in solution and undergo violent collapse, that produce localized high temperatures and pressures. Based on the literature, both types of cavitation could be responsible for cell death via a mechanical pathway, while only the inertial cavitation could result in sonochemically induced cell death [5,6]. The presence of certain drugs in sonication conditions could greatly enhance cell death. Studies showed that these effects occur not via increasing the cellular susceptibility to the sonomechanical forces but rather due to the sonochemical reactions [7–9]. These synergistic effects of drugs, which are also known as sonosensitizers, and ultrasound are termed as sonodynamic therapy (SDT). Porphyrins or porphyrin-like compounds, such as protoporphyrin IX (PpIX), are the agents of choice in most SDT experiments because they have specific accumulation in diseased tissues, such as tumors [10]. PpIX could significantly enhance the cell damaging

http://dx.doi.org/10.1016/j.ultsonch.2015.04.024 1350-4177/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: H. Xu et al., The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.04.024

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H. Xu et al. / Ultrasonics Sonochemistry xxx (2015) xxx–xxx

effects of ultrasonic exposure on different cancer cell lines in vitro [11]. In addition, pretreatment of cells with antioxidant agents such as N-acetylcysteine (NAC), cysteamine, mannitol and catalase, greatly reduce the occurrence of cell death [10,12,13]. These prior studies indicate that the sonochemical activation of PpIX and the subsequent production of reactive oxygen species (ROS) may play a pivotal role in SDT-induced cell death. However, the exact mechanism involved in the sonochemical effects is still not clear. To simulate the sonochemical effects, aqueous solutions of sonosensitizers irradiated by ultrasound were investigated in different studies and the ROS produced were identified using different methods [12,14]. Riesz et al. and Umemura et al. studied the effect of sonication on aqueous hematoporphyrin solutions. They used sensitive reagents capable of trapping a specific free radical and then detected them using electron spin resonance [7,15]. Wang et al. studied the effects of ultrasonic irradiation on aqueous solutions of chlorophyllin metal complexes using oxidation-extraction spectrometry [16]. They showed that the 1,5-diphenyl carbazide was oxidized by the ROS into 1,5-diphenyl carbazone, which displayed a visible absorption at approximately 563 nm. It has been shown that free radicals produced by ultrasonic irradiation could activate porphyrins and result in their decomposition [12,17]. Porphyrins themselves are fluorescent chemicals and can be detected using a fluorescence spectrophotometer. Thus, the fluorescence intensity of porphyrin itself can be indicative of the formation of free radicals without the addition of new trapping agents. In the present study, aqueous PpIX solutions were irradiated by ultrasound. The inertial cavitation was evaluated using terephthalic acid dosimetry. The PpIX fluorescence intensity as well as the fluorescence and absorption spectra were acquired before and after sonication. In addition, factors, such as the initial PpIX concentration, the duration of ultrasonic irradiation and the ultrasound intensity and duty cycle were evaluated to determine their influence on PpIX decomposition. Finally, several scavengers were used to determine the types of ROS involved in.

2. Materials and methods 2.1. Experimental apparatus The apparatus used for ultrasound exposure in this study is shown in Fig. 1. The experimental setup was composed mainly of an ultrasound transducer and a polypropylene test tube. The ultrasonic system including a transducer (diameter: 3.5 cm, resonance frequency: 1.0 MHz), a wave form generator and a power amplifier, was assembled in house by the Harbin Institute of Technology (Harbin, China). The transducer and the test tube were immersed in a water bath filled with degassed water at room temperature 25 °C. The transducer was placed at the bottom of the water bath, and the tube was placed 20 cm above the transducer. The internal surface of the experimental bath was padded with ultrasound-absorbing foam to minimize the standing wave formation reflected from the lateral wall. The intensity of the ultrasound was measured using a needle hydrophone (1 mm in diameter) (Onda Corp., Sunnyvale, CA, USA). The hydrophone was placed 20 cm away from the center of the transducer surface where the test tube was sonicated in the water bath. The detected signal was registered on a digital oscilloscope (Tektronix, Beaverton, OR, USA). The acoustic intensity (I) can be calculated using the equation I = P2/qv, where P is the sound pressure that represents the local pressure deviation from the average ambient pressure caused by a sound wave, q is the density of the medium, and v is the speed of sound in the medium. The spatial average value was evaluated using a three-dimensional micromanipulator scanning system, and the temporal

Fig. 1. Schematic diagram of the sonication setup.

average value was analyzed as a function of the sonication duration. An acoustic intensity in the range of 0 to 1.5 W/cm2 (ISATA, spatial average time average intensity) was used according to the different experimental demands. 2.2. Ultrasonic irradiation protocol Ultrasound intensities ranging from 0 to 1.5 W/cm2 were applied to determine the threshold intensity levels for the PpIX decomposition and the onset of inertial cavitation. To determine the effects of ultrasonic duty cycle (DC) on PpIX decomposition, DCs from 10% to 100% were investigated. In most experiments, the ultrasound with 1.2 W/cm2 intensity and 20% DC was employed. The pulse repetition frequency (PRF) was 100 Hz in burst ultrasound. A total of 4 ml air-saturated reaction solution was prepared via air bubbling for 3 min before sonication in the dark. The temperature of the solution was measured after ultrasound exposure. Thermal effects were excluded as no significant variation in temperature (±2 °C) was detected after sonication either in the tone-burst or the continuous mode. 2.3. Cell culture and measurement of cell death by Annexin V-FITC and PI staining Human THP-1 cells (ATCC, Rockefeller, MD, USA) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Before treatment, the cell culture medium was replaced with serum free RPMI 1640 medium and the cells were placed in the test tube. The PpIX was given by incubating cells with 5-aminolevulinic acid (1 mM) which could form PpIX in the cells. The ultrasound parameter was 0.9 W/cm2, 10% DC and 5 min irradiation. 4 h after treatment, the cell death was determined by staining cells with Annexin V-FITC and counterstaining with propidium iodide (PI). Annexin V-FITC binds to phosphatidylserine (PS) on the outer leaflet of the plasma membrane while PI is excluded by cells with intact membranes which could used to detect the apoptotic cells. Briefly, 0.5  106 cells were washed twice with PBS and stained with 5 ll of Annexin V-FITC and 10 ll of PI (BD PharMingen, San Diego, CA, USA) in 1  binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) for 20 min at 4 °C in the dark. Apoptotic cell ratio was analyzed using FACScan flow cytometer (BD Biosciences) and Cell Quest analysis software.

Please cite this article in press as: H. Xu et al., The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.04.024

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solution pH. Then, PpIX solutions at different concentrations (1– 10 lM) were exposed to ultrasonic irradiation. The fluorescence intensity of PpIX was measured using a fluorescence spectrophotometer before and immediately after sonication. The fluorescence and absorption spectra were also scanned using a fluorescence spectrophotometer and a UV spectrometer (UNICAM 300, Thermo Spectronic, USA), respectively. The undecomposed PpIX ratio after sonication is defined as the ratio of the fluorescence intensity after exposure (F1) to the fluorescence intensity before exposure (F0). The decomposition ratio of PpIX is defined as the remainder of the undecomposed PpIX from 100% (1 F1/F0). 2.5. Terephthalic acid dosimetry A stock solution of 2 mM terephthalic acid (TA) (Sigma–Aldrich) was prepared as previously described, and kept in the dark at 4 °C [18]. TA is initially non-fluorescent, which form fluorescent 2-hydroxyterephthalate ions (HTA) on reaction with hydroxyl radicals. This reaction provides a very sensitive method for the estimation of cavitation activity in ultrasound fields [19]. Before and immediately after sonication, the fluorescence intensity of HTA was detected using a fluorescence spectrophotometer at 323 nm excitation and 424 nm emission. 2.6. Reactive oxygen species scavenger studies

Fig. 2. Apoptosis induced by ultrasound and PpIX treatment. Data were represented from at least three independent experiments. ⁄⁄denotes differences from the control group at p < 0.01.

Mannitol, methanol, ethanol, acetone (hydroxyl radical scavengers), catalase (hydrogen peroxide removal), superoxide dismutase (SOD, superoxide anion scavenger), sodium azide (NaN3, singlet oxygen quencher) and histidine (both hydroxyl radical and singlet oxygen quencher) were all purchased from Sigma– Aldrich. The scavengers were added to the PpIX solutions immediately before ultrasound exposure. The concentrations of the enzymatic scavengers used were 100 U for SOD and 1 KU for catalase. Sodium azide was used at a concentration of 10 mM. Histidine was applied from 1 mM to 100 mM. The concentrations of mannitol, acetone, methanol and ethanol were in the range from 5 mM to 500 mM. 2.7. Statistical analysis

2.4. Protoporphyrin IX decomposition PpIX with a purity P95% was purchased from Sigma-Aldrich Company (St. Louis, MO, USA). The material was supplied as a powder, and was prepared in 10 mM NaOH solution and stored in the dark at 4 °C. Before sonication, the stock solution was diluted with phosphate-buffered saline (PBS, pH = 7.4) to final concentrations of 1 lM–10 lM. The addition of PpIX did not cause any change in the

All data were presented as means ± standard deviation (SD) from at least five independent experiments. A one-way analysis of variance (ANOVA) was used to analyze differences among groups. Correlation between PpIX decomposition ratio and HTA fluorescence intensity at different sonication durations were assessed by Pearson correlation analysis. All statistical analyses were performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Differences with p < 0.05 were considered statistically significant.

Fig. 3. Thresholds of ultrasound intensity for PpIX decomposition (a) and onset of inertial cavitation. (b) The correlation between the PpIX decomposition ratio and the HTA fluorescence intensity at different sonication durations (0–10 min). (c) Values obtained from five independent experiments.

Please cite this article in press as: H. Xu et al., The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.04.024

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fluorescence intensity at different sonication durations from 0 to 10 min (Fig. 3c). 3.3. Effects of protoporphyrin IX concentrations on the PpIX decomposition The effects of initial PpIX concentrations (1–10 lM) on PpIX decomposition were investigated. As shown in Fig. 4a, the ratio of PpIX decomposition decreased with an increase in PpIX concentration. However, the amount of decomposed PpIX, which was calculated using the decomposition ratio multiplying the initial amount of PpIX, increased significantly from 3.09 ± 0.24 nmol to 4.82 ± 0.15 nmol and then to 7.29 ± 0.42 nmol (p < 0.001) when the PpIX concentration was increased from 1 lM to 2 lM and then to 5 lM (Fig. 4b). The amount of decomposed PpIX in the 10 lM group was not significantly different from that of the 5 lM group (6.54 ± 0.93 nmol versus 7.29 ± 0.42 nmol, p > 0.05). 3.4. Effects of ultrasonic duty cycles on PpIX decomposition

Fig. 4. The decomposition curves of PpIX during sonication (a) and the amount of decomposed PpIX after 10 min sonication (b) as a function of different PpIX concentrations. Data were represented as five independent experiments. ⁄⁄⁄ p < 0.001, NS: not significant.

The effects of ultrasonic DCs (10–100%) on PpIX decomposition were also studied. As shown in Fig. 5a, after ultrasound exposure for one minute, the decomposition ratio at 100% DC (42.66 ± 4.56%) was significantly higher than that at 50% DC (17.99 ± 1.39%), 20% DC (8.05 ± 0.51%) and 10% DC (5.00 ± 0.52%) (p < 0.001). Then, the sonication durations were compensated for tone-burst ultrasound at different duty cycles, 10 min for 10% DC, 5 min for 20% DC and 2 min for 50% DC. With the compensation of sonication durations (Fig. 5b), the decomposition ratio at 10% DC (42.96 ± 1.84%) was not different from those at 20% DC

3. Results 3.1. Synergistic cell death induced by ultrasound and protoporphyrin IX To test the ability of ultrasound and PpIX to induce synergistic cell death in our apparatus, human THP-1 cell line was used. As presented in Fig. 2, the apoptotic cell ratio in the ultrasound and PpIX-treated group was significantly higher than that in the control group (24.81 ± 3.89% versus 9.86 ± 1.94%, p < 0.01). The group treated with PpIX or ultrasound alone did not show any cytotoxic effects. To further simulate the sonochemical effects, the aqueous PpIX solution was irradiated with ultrasound, and the process was studied further. 3.2. Correlation between protoporphyrin IX decomposition and inertial cavitation To determine the thresholds of ultrasound intensity for PpIX decomposition and the onset of inertial cavitation, the decomposition ratio of PpIX and the HTA fluorescence intensity were measured after sonication at intensities from 0 to 1.5 W/cm2. As shown in Fig. 3a and b, the PpIX decomposition ratio and the HTA fluorescence intensity increased with ultrasound intensities from 0.6 to 1.5 W/cm2. The interception point between the dotted line and the x-axis was conceived as the threshold [20]. When the intensity was 0.3 W/cm2 or less, there was no PpIX decomposition or inertial cavitation. The threshold intensities for PpIX decomposition and onset of inertial cavitation were almost at the same level of 0.4 W/cm2. There was a positive correlation (R = 0.9874, p < 0.05) between the PpIX decomposition ratio and the HTA

Fig. 5. The PpIX decomposition ratio with one minute exposure (a) and with compensated sonication durations (b) at different duty cycles. Error bars show the standard deviation of the mean values obtained from five independent experiments. ⁄⁄⁄denotes differences from 10%, 20% and 50% duty cycle at p < 0.001.

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the UV absorption of these five peaks decreased but showed no changes in the spectral positions or shapes of the absorbance bands. 3.6. Effects of reactive oxygen species scavengers on protoporphyrin IX decomposition To identify the ROS involved in the ultrasonic activation of PpIX, aqueous PpIX solutions with or without ROS scavengers were exposed to ultrasound for 10 min. As shown in Fig. 8a, the relative PpIX fluorescence intensity in the presence of SOD (22.05 ± 4.04), catalase (27.65 ± 2.14) and sodium azide (30.38 ± 3.60) were not different from that of the ultrasound alone group (22.75 ± 5.98) (p > 0.05). However, the addition of histidine (Fig. 8b) at concentrations from 1 mM (54.48 ± 4.15) to 100 mM (94.10 ± 1.86) significantly inhibited PpIX decomposition (p < 0.001). The addition of mannitol from 5 mM (59.92 ± 3.35) to 500 mM (88.87 ± 3.70), acetone from 5 mM (79.44 ± 3.63) to 500 mM (80.39 ± 7.26), methanol from 5 mM (87.76 ± 2.12) to 500 mM (96.70 ± 1.91) and ethanol from 5 mM (89.23 ± 1.66) to 500 mM (98.18 ± 0.91) all significantly decreased the amount of PpIX decomposition (p < 0.001) (Fig. 8c–f). The PpIX emission spectrum was also studied in the presence of mannitol (Fig. 9). The decay of peak fluorescence was partially inhibited compared to that shown in Fig. 6a. There was no newly formed peaks or spectral shifts. Similar spectroscopic changes were acquired in the presence of histidine, acetone, methanol and ethanol (data not shown). Fig. 6. Fluorescence emission spectrum (a) and excitation spectrum (b) of PpIX solutions after sonication.

4. Discussion (38.63 ± 2.99%), 50% DC (38.31 ± 4.85%) or 100% DC (42.66 ± 4.56%) (p > 0.05). 3.5. Spectroscopic changes of protoporphyrin IX solutions before and after sonication To study the spectroscopic changes of the PpIX solutions after sonication, the fluorescence emission and excitation spectra as well as the absorption spectra were recorded. As shown in Fig. 6a, the fluorescence peak intensity of PpIX emission spectrum decayed significantly as a function of sonication duration. There was no spectral shift or newly formed peak after sonication. The excitation spectrum of the PpIX solutions showed similar spectroscopic changes after sonication (Fig. 6b). As shown in Fig. 7, the absorption spectrum of the PpIX solutions measured using a UV spectrometer displayed five distinct peaks at 397, 505, 537, 567, and 630 nm, with a maximum peak at 397 nm. After sonication,

Fig. 7. Absorption spectrum of PpIX solutions before and after ultrasound irradiation.

The synergistic cell killing effects of ultrasound and porphyrin derivatives have been demonstrated in cell studies and in animal models [9,20–22]. Those studies indicated that the synergistic action may be due to inertial cavitation-induced sonomechanical and sonochemical effects [23]. In this study, ultrasound together with PpIX was proved to cause synergistic cytotoxic effects. We have characterized the ultrasound activation of aqueous PpIX solutions and identified the free radicals participating in this process. Our results showed that hydroxyl radicals generated from inertial cavitation were involved in the PpIX activation. Inertial cavitation represents the growth and rapid collapse of bubbles in a liquid irradiated with ultrasound. It is a unique source of energy for driving chemical reactions [24,25]. In the present study, inertial cavitation was indirectly evaluated using TA dosimetry. The results showed that the threshold of ultrasound intensity for inertial cavitation was at the same level as for PpIX decomposition (Fig. 3a and b), and that the PpIX decomposition ratio was significantly correlated with the level of inertial cavitation at various sonication durations (Fig. 3c). These results suggest that inertial cavitation is essential for the sonochemical activation of PpIX [26,27]. The synergistic cytotoxic effects of ultrasound and certain drugs increased with the drug concentration and ultrasound intensity over a specific range [7]. The results in this study showed that when the concentration of PpIX was in the range of 1–5 lM, ultrasound-induced PpIX decomposition increased with the PpIX concentration. This could be explained by the study of Torii et al. [28]. It is suggested that for a certain ultrasonic irradiation system, when the generation of hydroxyl radicals is constant, hydroxyl radical induced reaction rate increases with the initial concentration of the solutes. At high concentration level (10 lM), however, ultrasound did not induce higher amount of PpIX decomposition (Fig. 4b). These results were in agreement with previous reports [29]. It is assumed that when the initial concentration of PpIX is low, for example less than 5 lM in our study, the diffusion of

Please cite this article in press as: H. Xu et al., The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.04.024

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Fig. 8. Effects of reactive oxygen species (ROS) scavengers on the PpIX decomposition. PpIX aqueous solutions were sonicated in the presence of superoxide dismutase (SOD, 100 U), catalase (1 KU), and sodium azide (NaN3, 10 mM) (a). PpIX decomposition in the presence of histidine (1–100 mM) (b). PpIX decomposition in the presence of mannitol, acetone, methanol and ethanol (5–500 mM) (c–f). Values were recorded based on five independent experiments. NS: not significant from the ultrasound alone group. ⁄⁄⁄denotes differences from the ultrasound alone group at p < 0.001.

PpIX into the interfacial cavitation region is assumed to increase with the amount of degradation. However, when the initial PpIX concentration is high, the diffusion of the PpIX is limited, and the production of free radicals determines the degradation [29]. In addition, a higher PpIX concentration could suppresses acoustic bubble formation by increasing the intermolecular tension on the bubble surface [6,30]. In this case, the accumulation of PpIX at 5 lM concentration might reach a plateau at the bubble surface and a higher concentration does not guarantee more PpIX decomposition. The effects of ultrasound DCs on PpIX decomposition were further investigated. Ebrahiminia et al. used iodide dosimetry to evaluate the inertial cavitation activity and showed that continuous ultrasound produced more sonochemical effects than burst ultrasound [31]. Our results also showed that with the same sonication duration, continuous ultrasound induced more PpIX decomposition than burst ultrasound (Fig. 5a). However, when the sonication durations at different DCs were compensated, there was no difference in the PpIX decomposition ratio as shown in Fig. 5b. This is different from the studies of Ebrahiminia et al. and Barati et al., who showed that continuous ultrasound also induced higher

cavitation activities than 20% DC with a compensated duration [27,31]. One explanation is that the sonication durations in our study were relatively short (1–10 min), whereas, in the studies of Ebrahiminia et al. and Barati et al., the durations were in the range of 20–100 min. During long sonication durations, the gas content is usually decreased. Thus, the relatively short durations used in our study do not contribute to the differences in the PpIX decomposition induced by continuous ultrasound and burst ultrasound with compensated durations. Hence, it is the total cumulative energy of the ultrasound controls the amount of decomposition. The spectroscopic changes in the PpIX fluorescence and absorption spectra were also measured before and after sonication. The fluorescence peaks of PpIX emission and excitation spectra as well as the absorption spectrum decayed after sonication (Figs. 6 and 7). Furthermore, we observed no spectral shift or newly formed peak in the fluorescence or absorption spectra. These spectroscopic changes indicate that the sonochemical activation of PpIX is simply a decomposition reaction and that there is no oxidative modification on the porphyrin rings. The PpIX decomposition induced by ultrasound is different from that induced by light irradiation during photodynamic therapy (PDT). In light activation of PpIX, the

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of PpIX as a process called sonobleaching, which is different from photobleaching in PDT. However, there are limitations in our study. The chemical effects of ultrasound on the PpIX decomposition alone cannot fully explain the results of in vivo works. The conditions in which ultrasound could produce inertial cavitation in in vitro media could not be reproduced in vivo due to a high tissue viscosity [37,38]. Additionally, inertial cavitation is sometimes dispensable for ultrasound-related treatments when low-intensity ultrasound is applied. Our work only simulates the sonochemical activation of PpIX in vitro. Further in vitro and in vivo studies still need to be performed to elucidate the sonobleaching phenomenon.

Fig. 9. Fluorescence emission spectrum of PpIX solutions during sonication in the presence of mannitol.

decrease of PpIX fluorescence intensity and the build-up of a photoproduct absorbing at 670 nm can be observed, which is a process called photobleaching [32–34]. These spectroscopic changes in PDT were demonstrated to be caused by the reactions between singlet oxygen and PpIX [32]. Therefore, in our study, the absence of new peak build-up excludes the major role of singlet oxygen in ultrasound activation of PpIX. The difference in the spectroscopic changes in the PpIX activation by ultrasound and light indicates different chemical reactions. In this study, ultrasound-induced PpIX decomposition was significantly inhibited by mannitol, histidine, acetone, methanol and ethanol, but it was not inhibited by NaN3, SOD or catalase (Fig. 8). Because the solutions were air saturated, the hydrogen atoms formed from inertial cavitation were immediately scavenged by oxygen [5]. Oxygen atoms produced inside the acoustic bubbles also could result in PpIX decomposition, however, the large inhibitory effects of the hydroxyl radical scavengers exclude oxygen as a major role [35]. Hydroxyl radicals were mainly responsible for the sonochemical activation of PpIX. It has been shown that sonochemical reactions take place in three different regions: the first is the high-temperature region of the collapsing gas bubbles, the second is the interfacial region between the hot gas and the bulk liquids, and the third region is the bulk of the solution at ambient temperature where the radicals that have escaped from the hot zones react with organic solutes [3,8]. Because PpIX possesses a low hydrophilicity, we propose that a portion of the PpIX decomposition occurs in the second region and the major portion occurs in the third region. The pyrolysis reaction of PpIX inside the collapsing gas bubble might be low. Accordantly, our scavenger studies also showed that the decomposition reactions did not seem to occur under a high concentration of ethanol and methanol. However, we cannot exclude the possibility that pyrolysis of protoporphyrin IX might occur in the immediate vicinity of the collapsing cavitation bubbles. Hydroxyl radicals formed from water sonolysis are highly reactive. Thus, we proposed that hydroxyl radicals decompose PpIX and produce sonosensitizer-derived free radicals such as peroxyl and alkoxyl radicals, which are more likely to reach critical cellular sites [8]. The fluorescence peak decay in the PpIX emission spectrum was partially attenuated in the presence of hydroxyl radical scavengers, and no newly formed peaks were discovered (Fig. 9). Therefore, when hydroxyl radicals were scavenged, singlet oxygen did not seem to participate in the ultrasound-induced PpIX decomposition. Nevertheless, we cannot exclude the existence of singlet oxygen, which is also indicated in other studies [7,21,36]. The amount of singlet oxygen might not be comparable to that of hydroxyl radicals, and thus, the decomposing effect by singlet oxygen was difficult to differentiate in our sonication system. Hydroxyl radicals due to inertial cavitation are involved in the ultrasound activation

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Please cite this article in press as: H. Xu et al., The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, Ultrason. Sonochem. (2015), http://dx.doi.org/10.1016/j.ultsonch.2015.04.024