This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.
In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 µs), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.
High intensity focused ultrasound (HIFU), or focused ultrasound surgery (FUS), is a non-ionizing and non-invasive technique that is used for the thermal ablation of subcutaneous tissue1. The main use of HIFU is in the treatment of soft tissue tumors2, but it is starting to be used for other applications, such as treating bone tumors3 or neurological conditions4. There are two main factors that limit the widespread use of HIFU in the clinic: firstly, difficulties in treatment guidance and secondly, long treatment times5. The combination of HIFU, pulsed laser illumination, and plasmonic gold nanorods described by this method could provide a way to overcome the current limitations for HIFU6.
During HIFU exposures, the dominant mechanism of tissue ablation is thermal damage. However, cavitation activity can also play a role8. Cavitation activity that occurs during HIFU exposures can consist of both mechanically and/or thermally mediated cavitation. Mechanically mediated cavitation is generally referred to as acoustic cavitation7, which is further subcategorized as bubbles undergoing either non-inertial or inertial9 behavior. Thermally mediated cavitation is from the formation of gas pockets, through ex-solution or vaporization, and is commonly referred to as 'boiling'10. Cavitation activity, most commonly inertial cavitation, has been shown to enhance the thermal heating rates achievable through HIFU exposures11 and thus help address one of its key limitations. However, the formation and activity of cavitation during HIFU exposures can be unpredictable and lead to negative effects such as over-treated regions, or asymmetrical thermal ablation12. In order to control cavitation activity during HIFU exposures, the introduction of external nuclei has been investigated. These can take the form of microbubbles13, phase-shift nanoemulsions14 or plasmonic nanoparticles15. Both microbubbles and nanoemulsions have been shown to improve signal-to-noise for imaging and enhanced thermal ablations. However, their transient nature means they have limited functionality over repeated HIFU exposures. Monitoring of cavitation activity during HIFU exposures is done using either active or passive cavitation detection (ACD or PCD, respectively). PCD is a favored technique for cavitation detection, as it can be performed concurrently with HIFU exposures and provides spectral content information. This spectral content can then be further analyzed to help identify the type of cavitation activity occurring16. Broadband acoustic emissions are used, since these emissions are unique to the presence of inertial cavitation10 and are linked to enhanced HIFU heating11.
Photoacoustic imaging (PAI) is an emerging clinical imaging technique17, which combines the spectral selectivity of pulsed laser excitation with the high resolution of ultrasound imaging18. It has previously been used to guide HIFU exposures19, but this imaging technique is limited by the penetration depth of laser light. Plasmonic gold nanoparticles can be used to act as 'contrast agents' increasing the local absorption of laser light and subsequently the amplitude of photoacoustic emissions20. For sufficiently high laser fluences, it is possible to cause the generation of microscopic vapor bubbles that can be used for highly localized imaging21. However, these exposure levels typically exceed the maximum permissible exposure limit for the use of laser light in humans22, and thus have limited use. The method employed in this study has previously shown that by simultaneously exposing the plasmonic nanoparticles to both laser illumination and HIFU, the laser fluence and acoustic pressures needed to nucleate these small vapor bubbles is dramatically reduced, and the signal-to-noise ratio for imaging is increased23. A method is described here for combining plasmonic nanoparticles with both laser and HIFU exposures for a highly controllable technique for the nucleation and activity of vapor bubbles.
1. Tissue Mimicking Phantom Manufacture
NOTE: An in-depth analysis of the acoustic properties of the optically transparent tissue-mimicking phantom used for all exposures in this study can be found in Choi, et al.24
NOTE: Each phantom mold contains approximately 50 mL of solution, and for each batch a total of five molds are filled. Thus, a total of 250 mL of phantom solution is prepared.
2. Calibration of HIFU Transducers Free Field Acoustic Pressure
NOTE: This section of the protocol is not necessary before every lesioning/imaging experiment. It is a calibration procedure to be performed at regular intervals to ensure acoustic output of the system is correct.
3. Configuring Experimental Apparatus for Both Pulsed and Continuous Wave Studies
4. Cavitation Threshold Detection from Pulsed HIFU Exposures
NOTE: The following procedure is the same for phantoms with or without nanoparticles, and should be repeated three times.
5. Thermal Denaturation from Continuous Wave HIFU Exposures
NOTE: The following procedure is the same for phantoms with or without nanoparticles and were repeated three times.
Cavitation detection from pulsed HIFU exposures
The passive cavitation detection system recorded the voltage/time data for the range of HIFU and laser exposures in both phantoms with and without nanoparticles. Figure 2 shows the representative results for a range of exposures. The time scales on these plots are truncated to highlight the regions where broadband acoustic emissions would be expected, due to the time of flight of these emissions. Figure 2 demonstrates that it is only when there is a combination of nanoparticles, HIFU exposure and laser illumination that broadband emissions are detected. However, this is still a threshold phenomenon, as at the lower acoustic pressure for Figure 2h broadband emissions were not detected. The duration of these emissions typically correspond to the length of the HIFU exposure, which was around 10 µs in this study.
Thermal denaturation from a CW HIFU exposure
Figure 3 shows a series of frames acquired from the universal serial bus (USB) camera during a single HIFU exposure with laser illumination, for the three different exposures types (with/without laser illumination and/or nanoparticles). This figure shows an example of the formation of thermal lesions in the gel phantoms for each of these conditions. In this view the HIFU exposure occurs from left to right. For the example shown in Figure 3 the peak negative pressure was 2.53 MPa, which was the upper edge of what was used in this study.
Recording inertial cavitation dose (ICD) from CW HIFU exposures
Figure 4 shows representative results from the calculation of ICD recorded during CW HIFU exposures. This data was post processed from the emissions recorded by the PCD system during the exposure. Figures 4a, 4c, and 4e show that at a lower peak negative pressure, no broadband emissions were detected, where Figures 4b, d, and f show that ICD was recorded throughout the exposure. The highest ICD signals were observed during the exposure in a gel containing nanoparticles with both HIFU and laser exposures (Figure 4f).
Figure 1. A schematic representation of the experimental apparatus used in this study. For clarity, the USB microscope and light source are omitted, but the view region is illustrated by a blue dashed box. CNC – Computer numerical control, AuNR – Gold nanorods. Figure adapted from McLaughlan et al. (2017)6. Please click here to view a larger version of this figure.
Figure 2. An example of the voltage traces recorded with the passive cavitation detection system during short HIFU exposures, with/without simultaneous laser illumination. When used, the laser fluence was 2.1 mJ/cm2 with a peak negative pressure of (a-c) 3.0, (d-f) 2.13 and (g-i) 1.43 MPa. LS – Laser, NR – nanoparticles. Please click here to view a larger version of this figure.
Figure 3. Individual frames at times 0, 5, 10 and 15 s during a HIFU exposure recorded by the USB microscope. The laser fluence was 3.4 mJ/cm2 and peak negative pressure of 2.53 MPa. Sequence (a) was with the laser exposure and in a phantom without nanoparticles, (b) is without laser exposure and in a phantom containing nanoparticles, and (c) has both laser illumination and a phantom containing nanoparticles. Please click here to view a larger version of this figure.
Figure 4. Calculated inertial cavitation dose (ICD) recorded during exposures (a, b, e, & f) with and (c & d) without laser illumination. Peak negative pressure was either (a, c, & e) 0.91 or (b, d, & f) 2.53 MPa. The phantom used in (a & b) did not contain any nanoparticles. Please click here to view a larger version of this figure.
This protocol is divided into four separate sections, describing the manufacture of the tissue-mimicking phantom through to the CW exposures in them to produce thermally generated denaturation. This denaturation of the phantoms simulates thermally generated coagulation necrosis experienced by soft tissue exposed to HIFU1. In their manufacture, it is important to ensure that the ratio of APS and TEMED is such that the process does not catalyze too quickly. As this process is exothermic, the faster this rate, the higher the temperature reached25 and thus could denature the BSA proteins prior to exposure. The ratio of APS to TEMED in this protocol has been set such that this should not occur, however the molds could be placed in ice water during the polymerizing of the gel to further minimize this possibility.
As this protocol focuses on the nucleation of cavitation through combining nanoparticles, laser illuminations and HIFU exposure, a critical step in the manufacture of the gel phantoms is to degas them under vacuum for a minimum of 30 min. Once exposed to HIFU (particularly CW exposures), even if a thermal lesion was not present, it is important to target a fresh location in the gel phantoms to avoid preexisting nuclei. When moving the phantom using the computer controlled translation system it is important to ensure that the depth of the HIFU focus (and thus aligned region) is kept consistent. This ensures that the HIFU pressure and laser fluence levels are uniform for each specific exposure parameter. For this protocol and after the initial placement of the phantom holder, it is then only translated in the vertical axis.
The temperature-sensitive tissue-mimicking gels are used widely by the HIFU research community25, as they provide a visual mechanism for monitoring the formation of a thermal lesion. This study was the first example of combining them with nanoparticles and demonstrating the enhancement provided to lesion formation through controlled cavitation activity. However, although they are classified as tissue-mimicking for their response to temperature, both their optical and acoustic attenuation are not. Due to the need to visualize the lesion formation in the gels, the phantoms are near transparent, with a slight yellow tint. As the laser fluence is adjusted to account for this, it does mean that the laser light illuminating the target region is collimated rather than diffusive as would be for normal tissue. Thus to allow for clinical translation multiple illumination sources would be needed to ensure enough fluence on the surface. Currently this work adheres to the guidelines22 for the safe use of lasers when exposed to skin. This would limit the maximum laser fluence achievable at depth; thus, this technique would initially be suited to treating superficial cancers such as breast, or head and neck. Furthermore, plasmonic nanoparticles targeted to surface receptors for these types of cancers could provide increased selectivity in treatments. However, even though this is a highly active area of research, no such particles are currently approved for clinical use.
The acoustic attenuation of the phantoms with nanoparticles was measured to be 0.7±0.2 dB/cm6, and, compared with the value for soft tissue of 3-4 dB/cm, it is significantly lower. Thus, the heating from HIFU exposures in these gels would be lower than would be observed in soft tissue. It has been demonstrated that addition of glass beads to the gel increases the attenuation levels similar to soft tissue25. However, in this application, this approach is not possible as these beads would act a nucleation sources for cavitation activity even in the absence of nanoparticles, and thus misrepresent the cavitation threshold. When comparing the heating efficiency for with the results from the study by Choi et al. (2013)25, thermal lesions were generated at peak pressure ranges of 14 – 23 MPa (it is not stated if this was peak positive or negative pressure). As this was performed at 1.1 MHz, the attenuation in the phantoms was lower than used in this study. Nevertheless, the nanoparticle-nucleated approach in this study was able to generate thermal lesions in these phantoms at pressures ranging from 1.19 to 3.19 MPa, thus demonstrating an increased efficiency over current methodologies.
Future testing for this methodology should be undertaken in an in vivo model to incorporate tumor reduction, tissue perfusion, molecular targeting of nanoparticles and relevant acoustic attenuation parameters.
The authors have nothing to disclose.
This work was supported by EPSRC grant EP/J021156/1. The author would like to acknowledge support from an early career Leverhulme fellowship (ECF-2013-247).
Single Element HIFU transducer | Sonic Concepts | H-102 | |
55dB Power Amplifier | E&I | A300 | |
Function Generator | Keysight Technologies | 33250A | |
Differential Membrane Hydrophone | Precision Acoustics Ltd | ||
TTL Pulse Generator | Quantum Composers | 9524 | |
Nd:YAG Pulse Laser | Continuum | Surelite I-10 | |
OPO Plus | Continuum | Surelite | |
Fibre Bundle | Thorlabs Inc | BF20LSMA01 | |
Energy Sensor | Thorlabs Inc | ES145C | |
Nanorods | Nanopartz | A12-40-850 | |
Broadband detector | Sonic Concepts | Y-102 | |
5 MHz high pass filter | Allen Avionics | ||
40dB preamplifier | Spectrum GmbH | SPA.1411 | |
14-bit data acquisition card | Spectrum GmbH | M4i.4420×8 | |
Deionised Filtered Water | MilliQ | ||
Acrylamide/Bis-acrylamide solution | Sigma Aldrich | A9927 | |
1 mol/L TRIS Buffer | Sigma Aldrich | T2694 | |
Ammonium Persulfate | Sigma Aldrich | A3678 | |
Bovine serum albumin | Sigma Aldrich | A7906 | |
TEMED | Sigma Aldrich | T9281 | |
3D printer | CEL-UK | Robox | |
3-axis positioning system | Zolix | ||
Digital Microscope | Dino-lite | AM4113TL | |
Water Tank | Muji | Acrylic Tank | |
Optical Components | Thorlabs Inc | Various | |
Optomechanical Components | Thorlabs Inc | Various | |
BNC Cables | RS | ||
Desktop PC | Custom Made | ||
Hotplate Stirrer | Fisher | ||
SBench6 | Spectrum GmbH | Measurement software |