Chapter 6 Passive Cavitation Detection During Skin Sonoporation Jeremy Robertson, Marie Squire and Sid Becker Abstract Passive cavitation detectors (PCDs) have been effectively employed in high-intensity focused ultrasound (HIFU) and cell sonoporation studies to monitor variations in inertial cavitation activity during the course of ultrasound application. As inertial cavitation is the mechanism responsible for many ultrasound induced bioeffects, this monitoring can provide valuable information in real time about the effectiveness of the ultrasound treatment. Despite the well-established benefits of employing PCD techniques in HIFU and cell sonoporation applications, little attempt has been made to utilize such techniques in the field of low-frequency skin sonoporation. This study presents an attempt to employ a confocal PCD system to monitor inertial cavitation activity during sonoporation in a Franz diffusion cell setup. To determine whether inertial cavitation activity was effectively monitored, the output of the PCD system was compared to the cavitation enhanced transport of caffeine through porcine skin. The correlation between caffeine transport enhancement and PCD response was poor relative to similar correlations presented in the literature. This result should not be seen as an indictment on the concept as the present study was only a first attempt at employing a confocal PCD in a skin sonoporation setup. The authors intend to refine their methodology and repeat the study. Keywords Cavitation · Sonoporation · Franz diffusion cell 6.1 Introduction The transdermal route is advantageous for drug delivery as it avoids the first pass metabolism effects which occur with oral delivery and the pain associated with intravenous injection. However, the skin acts as a natural barrier against most topical permeants. In skin sonoporation, a transducer produces a vibrating solid-fluid interface which creates an ultrasound field. This ultrasound field is used to temJ. Robertson (B) · M. Squire · S. Becker University of Canterbury, Christchurch, New Zealand e-mail: jeremy.robertson@pg.canterbury.ac.nz © Springer Nature Switzerland AG 2019 S. Gutschmidt et al. (eds.), IUTAM Symposium on Recent Advances in Moving Boundary Problems in Mechanics, IUTAM Bookseries 34, https://doi.org/10.1007/978-3-030-13720-5_6 63 64 J. Robertson et al. porarily enhance the skin’s permeability so that topical permeants may more easily diffuse. Five experimental parameters have been shown to influence skin permeability enhancement in skin sonoporation studies: the transducer frequency [1, 2], the ultrasound application time [3, 4], the distance from the transducer to the skin [3, 5], the chemical composition of the coupling fluid [6], and the ultrasound intensity [1, 7]. Each of these parameters can be independently controlled and maintained throughout ultrasound application and their influences on enhancement are well understood. Investigating the influences of these parameters has been, in part, motivated by the desire to optimize ultrasound enhancement [1, 3, 5]. However, such optimization cannot be achieved without first controlling the behavior of the mechanism that actually drives skin permeability increase: inertial cavitation [1, 2, 7]. Inertial cavitation activity can vary over the course of ultrasound application, even if all of the experimental parameters are held constant [8]. This is due to its dependence on coupling fluid temperature and the presence of cavitation nuclei in the coupling fluid, which can both vary during ultrasound application. In order to circumvent this variation and maintain a consistent amount of inertial cavitation activity in their high-intensity focused ultrasound (HIFU) tumor ablation setup, Hockham et al. [8] employed a novel technique. This involved a feedback loop that non-invasively monitored the inertial cavitation activity and then altered the transducer amplitude to mitigate any changes. Before attempting to apply the technique of Hockham et al. [8] to skin sonoporation, it is necessary to first address the question that motivated the present study: can the inertial cavitation activity in a skin sonoporation setup be effectively monitored during sonoporation? A system for monitoring inertial cavitation activity has already been effectively employed in several different ultrasound biophysics studies, including that of Hockham et al. [8]. This system is known as a passive cavitation detector (PCD). A PCD involves a hydrophone that is positioned in the coupling fluid and aligned with the ultrasound transducer beam in a confocal manner. The signal from this hydrophone is filtered to isolate a band of noise independent of the harmonic and sub-harmonic peaks in the frequency spectrum. The RMS value of this broadband noise emission has been shown to be indicative of the prevalence of inertial cavitation activity in the ultrasound beam [9]. In their study of HIFU induced blood-brain barrier opening, Tung et al. [10] used a PCD to investigate the pressure threshold for inertial cavitation in a blood vessel phantom. In the planar high-frequency cell sonoporation study by Hallow et al. [9], a confocal PCD system was used to monitor variations in inertial cavitation activity during ultrasound application. In that study, broadband noise was found to correlate with cellular bioeffects over a broad range of experimental conditions which lead the authors to advocate for a feedback system similar to the one employed by Hockham et al. [8]. Despite the effective use of PCDs in other ultrasound biophysics applications, there appear to be only three published studies in the field of skin sonoporation that have attempted to include a cavitation monitoring system. Tezel, Sens et al. [2] employed PCD techniques in their skin sonoporation study. However, the PCD data was captured prior to sonoporation instead of during, and although the hydrophone was confocally aligned with the transducer, no skin was present. A similar setup 6 Passive Cavitation Detection During Skin Sonoporation 65 was used in the study by Tezel and Mitragotri [11]. This study also employed PCD techniques, but not during skin sonoporation. In the study by Tang, Wang et al. [7] a transducer device was epoxied to the bottom of the apparatus. This was used as a PCD device despite the fact that it was not confocally aligned with the transducer in the coupling fluid. All three of these published studies reported encouraging results from their PCD systems, however, none of these systems captured noise emission data with a confocal hydrophone during skin sonoporation. A possible explanation for the limited use of PCDs in skin sonoporation setups can be found in the established geometry and materials used for Franz diffusion cells which are an integral part of most transdermal transport experiments [1, 3, 4, 12, 13]. Due to the small diameters of the donor chambers in these cells, usually around 15 mm [2], confocal positioning of a transducer and hydrophone in the coupling fluid is impractical. In order to facilitate a confocally aligned hydrophone, necessary for the correct implementation of a PCD, a partial redesign of the Franz diffusion cell is necessary. The purpose of this study was to assess the effectiveness with which a confocally aligned PCD was able to monitor inertial cavitation activity during skin sonoporation in a modified Franz diffusion cell. This was achieved by measuring the broadband noise emission over 10 min of ultrasound application at three different intensity levels. This noise emission was then compared with the transport of caffeine through the sonoporated skin to determine the correlation between transport enhancement and PCD response. 6.2 Materials and Methods 6.2.1 Modified Diffusion Cell Each of the 10 identical vertical Franz diffusion cells used in this study was made up of a donor and receiver chamber. The donor chamber had an inner diameter of 61 mm, an outer diameter of 65 mm, an aperture diameter of 9 mm, and a total volume of 96 mL. The receiver chamber had a volume of 3.2 mL and an aperture diameter of 9 mm. The donor, receiver and clamp geometries are shown in Fig. 6.1. The donor and receiver chambers were turned from solid polypropylene rods (Polystone, Dotmar EPP Pty Ltd, Christchurch, New Zealand) on a CNC lathe (Top-Turn CNC 406, Jashco Machine Manufacture Co. LTD, Taichung, Taiwan). Franz diffusion cells are usually made of glass, however, polypropylene was used in the present study due to its low cost and machinability. To form the sampling arm (Fig. 6.1), a section of carbon fiber tube (inner diameter 3.5 mm, outer diameter 6 mm) (Carbon Fiber Tube Pultruded, MAKERshop, Auckland, New Zealand) was glued to the polypropylene receiver with Loctite 401. Two pieces of carbon fiber tubing (inner diameter 2 mm, outer diameter 4 mm) were glued to the donor chamber in order to form the ports (Fig. 6.1) that enabled circulation of the coupling fluid for 66 J. Robertson et al. Fig. 6.1 Diffusion cell geometry (left) plan view, (middle) front view, and (right) isometric view temperature control (described in Sect. 2.4). The donor and receiver chambers were held together by a 3D printed clamp described in [14]. 6.2.2 Chemicals and Porcine Skin Caffeine powder (ReagantPlus) was purchased from Sigma-Aldrich (St Louis, MO). Phosphate buffered saline (PBS) (pH 7.4) was purchased from Thermofisher (Waltham, MA). The caffeine solution was prepared by dissolving the caffeine powder in room temperature PBS at a concentration of 0.5% w/v (5 g/L), as in the study by Sarheed and Abdul Rasool [15]. Porcine ears were obtained from Ashburton Meat Processors Ltd (Ashburton, New Zealand) immediately after slaughter. The ears were cleaned with cold tap water. The top 1 mm of the skin was removed from each ear using a dermatome (Dermatome 50 mm, Nouvag AG, Goldach, Switzerland). The dermatomed pieces of skin were flash frozen in liquid nitrogen using the technique described by Han and Das [16] then immediately transferred to a −20 °C freezer for storage. Prior to each set of experiments, the skin was removed from the freezer and thawed in a container of deionized water at room temperature. Each piece of skin was then visually assessed for uniform thickness and integrity before being mounted in a diffusion cell. 6.2.3 Ultrasound Generation and Intensity A low-frequency (20 kHz) ultrasound field was generated using a VC 505 ultrasound processor (Sonics and Materials Inc., Connecticut, USA). This unit was operated with a 13 mm diameter replaceable tip (Fig. 6.2). An application time of 10 min was used for all of the skin insonation experiments in this study. The transducer face was positioned 5 mm from the surface of the skin as this was the smallest distance 6 Passive Cavitation Detection During Skin Sonoporation 67 Fig. 6.2 System schematic. The solid lines represent the coupling fluid circuit. The dashed lines represent the signal inputs and outputs to and from the instruments in the coupling fluid Fig. 6.3 Cross section of the diffusion cell showing the positioning of the transducer, hydrophone, and thermocouple that allowed for a confocal hydrophone (shown in Fig. 6.3). The intensity of the ultrasound field was determined with the commonly used calorimetric method [3, 4, 13, 17–19]. This method is described in [14]. 68 J. Robertson et al. 6.2.4 Temperature Measurement and Control Continuous application of ultrasound at the intensities used in this study results in significant increases in coupling fluid temperature. A circulating method, described in [14], was used to mitigate this temperature increase during skin sonoporation. The temperature of the coupling fluid was measured with a thermocouple (Wire Type K thermocouple, Jaycar Electronics Pty Ltd, Auckland, New Zealand). The thermocouple was not positioned within the beam of the transducer, where it would have best represented the temperature of the coupling fluid near the skin surface, as this would have partially obscured the skin surface, and resulted in cavitation damage to the thermocouple tip. Instead the thermocouple was positioned outside of the transducer beam (Fig. 6.2). The difference in the temperatures recorded in these two positions was found to be less than 1.5 °C during sonoporation at 39.4 W/cm2 . 6.2.5 Passive Cavitation Detection The PCD hydrophone was positioned in the coupling fluid so that it was confocal with the ultrasound transducer (Fig. 6.2). This needle hydrophone (2.0 mm Needle Hydrophone, Precision Acoustics Ltd, Dorchester, Dorset, UK) had a sensitivity of −236.4 dBre1 V/µPa at the transducer driving frequency. The position of the hydrophone was kept consistent over all of the experimental repetitions by using an aluminum sleeve that was fixed relative to the transducer and diffusion cell. Between experimental repetitions, the hydrophone was taken out of this sleeve so that the diffusion cell could be switched out and the next one inserted. The method used to process the raw hydrophone data was described in [14]. Briefly, the hydrophone voltage data was filtered to isolate the broadband noise emission between 92.5 and 97.5 kHz. An RMS value of this filtered data was calculated every 1–2 s. All of the RMS values were then integrated over the 10 min of ultrasound application in order to calculate a single inertial cavitation dose value for each ultrasound application. Therefore, each inertial cavitation dose value represents the time-averaged broadband noise emission for a specific ultrasound application. 6.2.6 Chromatography Transdermal transport of caffeine was measured with a HPLC system (Ultimate 3000, Thermo Fisher Scientific, MA, USA). The solid phase consisted of a Poroshell 120 column (EC-C18, DKSH NZ Ltd, Palmerston North, New Zealand). This column was maintained at 40 °C during operation. The mobile phase consisted of 10% acetonitrile in water. The flow rate was 0.8 mL/min. The injection volume was 1 µL. 6 Passive Cavitation Detection During Skin Sonoporation 69 The retention time was 3.5 min and the reproducibility relative standard deviation was 1%. 6.2.7 Transdermal Transport Experiments Prior to the ultrasound application, a piece of skin was mounted onto each of the ten diffusion cells. The receiver fluid consisted of PBS while the donor chamber fluid was deionized water. One at a time, each diffusion cell was positioned along with the transducer, hydrophone and thermocouple. The temperature control system was then switched on to lower the coupling fluid (deionized water) to 10 °C. Continuous ultrasound was then applied for 10 min. Following sonoporation of each skin sample, fresh room temperature deionized water was added to the donor chamber to keep the skin hydrated while the other skin samples were sonoporated. After all ten of the skin samples had been individually sonoporated, the deionized water was removed from each of the donor chambers so that the caffeine solution could be applied. This solution was allowed to diffuse for a period of 20 h. During this time the donor chambers were covered with Parafilm (Bemis, WI, USA) to prevent evaporation of the fluid. This process was repeated for three different ultrasound intensities (23.8, 34.2, and 39.4 W/cm2 ). In addition to these experiments, two control cases were also investigated. For the first control case, the 10 diffusion cells were set up in the same manner described above, however, no ultrasound was applied. The coupling fluid was simply maintained at 10 °C for 10 min. For the second control case, no ultrasound was applied and the coupling fluid was maintained at 25 °C for 10 min. During each of the 30 ultrasound experiments, the temperature was kept between these two control temperatures by varying the voltage input to the temperature controller. 6.2.8 Physical Dosimeter Experiments The pitting of aluminum foil under insonation has previously been used to determine the influence of ultrasound parameters on inertial cavitation activity [4]. In the present study, aluminum foil was used as a physical dosimeter in order to demonstrate the influence of the temperature control method on the inertial cavitation activity at the skin aperture. Ten pieces of aluminum foil (Homebrand, Manukau, New Zealand) were insonated (at an intensity of 23.8 W/cm2 ) for 5 s with and without coupling fluid circulation. The number of pits in each of the 20 pieces of foil were then counted in order to demonstrate the influence of circulation. In these experiments, the coupling fluid in the donor chamber was deionized water at 10 ± 2 °C. 70 J. Robertson et al. 6.3 Results and Discussion The results presented in this study were collected in order to assess whether the inertial cavitation activity in a skin sonoporation setup can be effectively monitored during skin sonoporation. However, the present study represents the first time the temperature control system (described in Sect. 2.4) has been used during transdermal transport experiments. Therefore, it was necessary to first ensure that the coupling fluid circulation did not negatively impact the inertial cavitation activity at the skin aperture. This was achieved with physical dosimeter experiments. The mean number of pits after ultrasound application without coupling fluid circulation was 28.8. With coupling fluid circulation, the mean value was 26.3. These values (shown by the crosses in Fig. 6.4) indicate that coupling fluid circulation resulted in only a 9% decrease in inertial cavitation activity at the skin aperture (p  0.4). This small decrease in inertial cavitation is acceptable. In order to quantify the effects of ultrasound enhancement in this skin sonoporation study, it was necessary to measure the transdermal transport of caffeine through skin samples not exposed to ultrasound. Two such control cases were investigated (as described in Sect. 2.7). Following diffusion for 20 h, the mean and median receiver caffeine concentrations for the control at 10 °C were 31.6 mg/L and 27.6 mg/L respectively (Fig. 6.5). For the control at 25 °C, the mean and median receiver caffeine concentrations were 42.5 mg/L and 34.9 mg/L respectively. These values indicate that the coupling fluid temperature did have a small effect on skin permeability within this range. However, this effect is minor when compared to the effect of ultrasound exposure (Fig. 6.6). Ultrasound was applied to skin samples at three different intensities (as described in Sect. 2.7). Application at 23.8 W/cm2 resulted in mean and median receiver caffeine concentrations of 45.9 mg/L and 15.6 mg/L respectively (shown in Fig. 6.6). Application at 34.2 W/cm2 resulted in mean and median receiver caffeine concentrations of 111.4 mg/L and 95.2 mg/L respectively. Application at 39.4 W/cm2 resulted in mean and median receiver caffeine concentrations of 116.6 mg/L and 108.7 mg/L Fig. 6.4 The influence of coupling fluid circulation on the pitting of aluminum foil. The crosses within the boxes represent the mean values (n  10) 6 Passive Cavitation Detection During Skin Sonoporation 71 Fig. 6.5 Receiver chamber caffeine concentration after 10 min of exposure to 10 °C or 25 °C deionized water and 20 h of passive caffeine diffusion (n  10) Fig. 6.6 Receiver chamber concentration after 10 min of ultrasound exposure to various intensities. The coupling fluid temperature was maintained between 10 and 20 °C (n  10) respectively. When these values are compared to those from the two control cases, it is apparent that no enhancement was achieved at an intensity of 23.8 W/cm2 while ultrasound at 34.2 W/cm2 and 39.4 W/cm2 resulted in mean values that were, respectively, 2.6 (p < 0.05) and 2.7 (p < 0.05) times the mean of the 25 °C control case. This greater transport at the two higher intensities can be attributed to the higher degree of inertial cavitation achieved when the intensity is increased. Therefore, if the PCD setup worked as expected, the mean inertial cavitation dose should also have increased with increasing intensity. This trend is indeed apparent when inertial cavitation dose is plotted as a function of ultrasound intensity (Fig. 6.7). However, the analysis of the correlation between transdermal transport enhancement and PCD response must go further than this simple comparison of trends. In order to assess whether the PCD effectively monitored inertial cavitation activity during skin sonoporation, a direct comparison must be made between the inertial cavitation dose and the receiver caffeine concentration for all of the data across the three intensities. This comparison is shown in Fig. 6.8. The direct correlation between the inertial cavitation dose and receiver caffeine concentration values was poor. There is no clear separation between the intensity groups which was expected considering the clear intensity-concentration and 72 J. Robertson et al. Fig. 6.7 Inertial cavitation dose as a function of ultrasound intensity (n  9–10) Fig. 6.8 Receiver chamber caffeine concentration as a function of inertial cavitation dose intensity-dose trends shown in Figs. 6.6 and 6.7. There is also no clear trend within each intensity group. The poor correlation shown in Fig. 6.8 suggests that the PCD system used in this study did not represent the inertial cavitation behavior that occurred during skin sonoporation. However, this may be misleading. There are several features of the methodology that may have contributed to the poor correlation between caffeine transport and inertial cavitation dose. It is possible that the long caffeine residence time (20 h) caused the effects of the inertial cavitation activity on the skin permeability to be diminished by the effects of hydration which also acts to increase permeability over long periods of time. This issue could have been circumvented if periodic concentration samples had been taken during diffusion. Furthermore, the small molecular weight of caffeine (194 g/mol) may have limited the potential for permeability enhancement as only a small enhancement is possible with such a small drug. These points must be addressed, and the study repeated, if a definitive statement about the effectiveness of the PCD system is to be made. With these caveats in mind, the results of the present study should not be seen as an indictment on PCD systems in skin sonoporation. Nor should the results detract from the potential benefits of employing a functional cavitation feedback loop in a skin sonoporation setup. 6 Passive Cavitation Detection During Skin Sonoporation 73 6.4 Conclusions The purpose of this study was to assess the effectiveness with which a confocal PCD monitored the inertial cavitation activity within a Franz diffusion cell during skin sonoporation. If a PCD could be shown to perform in this context, that would enable the implementation of a feedback loop to control inertial cavitation activity. In order to facilitate an assessment of PCD performance, three different intensities were employed to create three distinctly different degrees of inertial cavitation activity. As expected, both receiver caffeine concentration and inertial cavitation dose increased with increasing intensity. However, a clear correlation between inertial cavitation dose and receiver caffeine concentration, which would have been indicative of an effective PCD, could not be obtained. The correlation was poor both between intensity groups and within intensity groups. The authors propose that using a larger permeant, and a shorter residence time will result in an improvement in the correlation. References 1. Tezel, A., et al.: Frequency dependence of sonophoresis. Pharm. Res. 18(12), 1694–1700 (2001) 2. Tezel, A., Sens, A., Mitragotri, S.: Investigations of the role of cavitation in low-frequency sonophoresis using acoustic spectroscopy. J. Pharm. Sci. 91(2), 444–453 (2002) 3. Terahara, T., et al.: Dependence of low-frequency sonophoresis on ultrasound parameters; distance of the horn and intensity. Int. J. Pharm. 235(1–2), 35–42 (2002) 4. Mitragotri, S., et al.: Determination of threshold energy dose for ultrasound-induced transdermal drug transport. J. Control. Release 63(1–2), 41–52 (2000) 5. Herwadkar, A., et al.: Low frequency sonophoresis mediated transdermal and intradermal delivery of ketoprofen. Int. J. Pharm. 423(2), 289–296 (2012) 6. Lavon, I., Grossman, N., Kost, J.: The nature of ultrasound–SLS synergism during enhanced transdermal transport. J. Control. Release 107(3), 484–494 (2005) 7. Tang, H., et al.: An investigation of the role of cavitation in low-frequency ultrasound-mediated transdermal drug transport. Pharm. Res. 19(8), 1160–1169 (2002) 8. Hockham, N., Coussios, C.C., Arora, M.: A real-time controller for sustaining thermally relevant acoustic cavitation during ultrasound therapy. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 57(12), 2685–2694 (2010) 9. Hallow, D.M., et al.: Measurement and correlation of acoustic cavitation with cellular bioeffects. Ultrasound Med. Biol. 32(7), 1111–1122 (2006) 10. Yao-Sheng, T., Choi, J.J., Konofagou, E.E.: Identifying the inertial cavitation pressure threshold and skull effects in a vessel phantom using focused ultrasound and microbubbles. AIP Conf. Proc. 1215(1), 186–189 (2010) 11. Tezel, A., Mitragotri, S.: Interactions of inertial cavitation bubbles with stratum corneum lipid bilayers during low-frequency sonophoresis. Biophys. J. 85(6), 3502–3512 (2003) 12. Smith, N., et al.: Ultrasound-mediated transdermal transport of insulin in vitro through human skin using novel transducer designs. Ultrasound Med. Biol. 29(2), 311–317 (2003) 13. Merino, G., et al.: Frequency and thermal effects on the enhancement of transdermal transport by sonophoresis. J. Control. Release 88(1), 85–94 (2003) 14. Robertson, J., Becker, S.: Influence of acoustic reflection on the inertial cavitation dose in a franz diffusion cell. Ultrasound Med. Biol. 44(5), 1100–1109 (2018) 15. Sarheed, O., Abdul Rasool, B.K.: Development of an optimised application protocol for sonophoretic transdermal delivery of a model hydrophilic drug. Open Biomed. Eng. J. 5, 14–24 (2011) 74 J. Robertson et al. 16. Han, T., Das, D.B.: Permeability enhancement for transdermal delivery of large molecule using low-frequency sonophoresis combined with microneedles. J. Pharm. Sci. 102(10), 3614–3622 (2013) 17. Mitragotri, S., et al.: Synergistic effect of low-frequency ultrasound and sodium lauryl sulfate on transdermal transport. J. Pharm. Sci. 89(7), 892–900 (2000) 18. Morimoto, Y., et al.: Elucidation of the transport pathway in hairless rat skin enhanced by low-frequency sonophoresis based on the solute–water transport relationship and confocal microscopy. J. Control. Release 103(3), 587–597 (2005) 19. Mutoh, M., et al.: Characterization of transdermal solute transport induced by low-frequency ultrasound in the hairless rat skin. J. Control. Release 92(1–2), 137–146 (2003)