Name: controllable nucleation of cavitation from plasmonic gold nanoparticles for enhancing high intensity focused ultrasound applications
Uploaded: 2018-10-05T13:30:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications at JoVE.com

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).

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.
<ol>
	<li>Add 148.2 mL (60% v/v) of deionized, filtered and degassed water to a 500 mL glass beaker and leave to equilibrate to room temperature. Add 75 mL of 40% (weight/volume) Acrylamide/Bis-acrylamide solution (30% v/v) to the glass beaker, followed by 25 mL of 1 M TRIS buffer, pH 8 (10% v/v), and 2.15 mL of 10% ammonium persulfate (APS; 0.86% v/v).</li>
	<li>Place the glass beaker inside a vacuum chamber that is situated on a magnetic stirrer plate, and place a 40 mm long polytetrafluoroethylene (PTFE) magnetic stirring bar inside the beaker. With a medium stirring speed (i.e., ensure good mixing without for formation of vortex in the water), slowly add 22.5 g (9% w/v) of bovine serum albumin (BSA) powder.</li>
	<li>Once all BSA has been added to the solution, close the vacuum chamber and turn on the vacuum pump. Maintain a vacuum of 80 mBar/H and continue stirring for a further 60 min, after which release the vacuum. At this point the solution should be clear with a slight yellow tint.</li>
	<li>The above methodology is the same for phantoms made both with and without nanoparticles. If nanoparticles are required, add 10 &#181;L (concentration of 1x108 np/mL) of nanorods that have a surface plasmon resonance (SPR) at 850 nm and a diameter of 40 nm.</li>
	<li>Finally, add 125 &#181;L of tetramethylethylenediamine (TEMED) to catalyze polymerization of the phantom. Wait a further 5 min to allow for mixing, then pour the phantom solution into 5 individual molds and wait 20 min to set. Once set, remove them from the holders and store in an airtight container until use. Use phantoms within 24 h of manufacture.</li>
</ol>
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.
<ol>
	<li>Fill an acrylic water tank (280x141x132 mm) with 4.5 L of deionized and degassed water. Mount the HIFU transducer on a fixed position post at one end of the tank, facing in. Parallel to this, mount a calibrated (performed by the National Physical Laboratories) membrane hydrophone onto a three-axis manual micrometer stage at the approximate focal point of the HIFU transducer (63 mm).</li>
	<li>Connect the HIFU transducer (geometric focus 63 mm) to the impedance matching circuit, then power amplifier (as shown in Figure 1). Then connect the membrane hydrophone directly to the data acquisition system, ensuring that a trigger signal is provided from the function generator connected to the power amplifier (Figure 1).</li>
	<li>Set the output voltage of the function generator to 30 mV, with a 10 cycle 3.3 MHz sine wave at a pulse repetition frequency of 100 Hz.</li>
	<li>Using the measurement software (see Table of Materials) to visualize the detected acoustic signal and the micrometer stage, position the detected acoustic pulse at the correct time of flight (42.5 &#181;m). Using only a single radial direction at a time on the micrometer stage, maximize the detected acoustic signal. Once confident this has been achieved, close the software and leave the membrane hydrophone in its current position.</li>
	<li>Vary the output voltage of the function generator from 20-400 mV in 20 mV increments. At each voltage level and using the MatLab acquisition software, record the hydrophone signals. Acquire 100 pulses at each level and convert from voltage data in pressure using the provided calibration data. Average the data and measure both the peak positive and negative values for all output voltage levels. This gives the calibration data for the free field peak negative pressure to be used for both the pulse and continues wave studies.</li>
</ol>
3. Configuring Experimental Apparatus for Both Pulsed and Continuous Wave Studies
<ol>
	<li>Fill an acrylic water tank (280x141x132 mm) with 4.5 L of deionized and degassed water. Mount the HIFU transducer and the co-aligned broadband hydrophone onto a three-axis manual micrometer stage. Then, fully submerge the transducer and hydrophone in the water tank. A schematic of this is shown in Figure 1.</li>
	<li>Connect the HIFU transducer to an impedance matching circuit, to enable it to be driven at its third harmonic (3.3 MHz). This circuit is connected directly to the output of a RF power amplifier. A digital function generator is connected to the input of the power amplifier, and programmed remotely.</li>
	<li>Prior to exposures in phantom material, use a calibrated differential membrane hydrophone to measure the peak negative pressure generated from this system for a given input voltage on the function generator as described in 2. Use these reference voltage values to set the required pressure level on the digital function generator.</li>
	<li>Connect the broadband hydrophone (geometric focus 63 mm) that is housed in the central aperture of the HIFU transducer directly to a 5 MHz high pass filter. Then connect it to a 14-bit data acquisition card (DAQ) via a 40 dB preamplifier. Ensure that the high pass filter is connected with the correct bias. 
	NOTE: This card was installed in a desktop PC and is used to control all hardware (examples this software can be found as supplementary files) and save data for off-line processing during this study.</li>
	<li>Connect a transistor-transistor logic (TTL) digital delay pulse generator with Bayonet Neill-Concelman (BNC) cables to both the pulsed laser system and function generator to ensure synchronization between these systems, which will ensure that the 7 ns laser pulse is coincident in the target region during the fourth rarefaction peak from the HIFU transducer.</li>
	<li>Using the method described in 1, omit the BSA and nanoparticles to make an alignment phantom, which is standard phantom material that contains a 1 mm spherical metallic target (a ball bearing). In order to achieve this, pour 25 mL of phantom material into a mold and add 62.5 &#181;l TEMED catalyst, then wait approximately 20 min to set. Then place the metallic target centrally in the phantom and add a further 25 mL of phantom solution followed by the 62.5 &#181;l TEMED catalyst and a further 20 min wait.</li>
	<li>Place the alignment phantom into the 3-D printed holder6, mount on an automated 3-D stage, and approximately position so that the metallic target is at the focal peak of the HIFU transducer.</li>
	<li>Using the HIFU transducer to send out a short duration 10 cycle burst (3 &#181;s) and the hydrophone to receive (connected directly to the DAQ card), the position relative to the alignment target is optimized through pulse-echo location. The real time detected signal will be displayed on the computer. Adjust the time of flight and signal amplitude using the manual micrometer stage that the HIFU transducer and hydrophone is mounted on. Once the time of flight is set to 85 &#181;s (a single round trip) and the signal amplitude has been maximized in both radial directions, this system will be aligned.</li>
	<li>Couple the optical energy from the optical parametric oscillator (OPO) pumped by the 532 nm nanosecond pulsed laser into the phantom using a 2 mm fiber bundle. Mount this fiber onto a second micrometer stage and position at an angle of 45&#730; from the acoustic axis in front of the phantom (Figure 1). The wavelength of the laser light is set to 680 nm to be visible for alignment. Once visible, position the laser illumination with the micrometer stage such that the alignment target is central in a 15 mm diameter laser spot.</li>
	<li>Position the 20-90x digital microscope (working distance 90 mm) and a white light source on opposite sides of the water tank perpendicular to the propagation plane of the HIFU transducer. The microscope is mounted on a small micrometer stage. Position it such that the metallic alignment targeted is central and in focus in its field of view (5x6 mm). 
	NOTE: After the above procedure is completed, all elements of this system (HIFU transducer, hydrophone, laser illumination and microscope) are now co-aligned to a specific location. The alignment phantom can now be replaced with the tissue-mimicking phantoms used for the study. As the phantom is mounted in a holder attached to a 3-D positioning system, different regions can be targeted whilst maintaining alignment.</li>
</ol>
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.
<ol>
	<li>Ensure that the PCD system is connected after being disconnected for the alignment procedure outlined in 3.8 and tune the laser wavelength to the SPR of the nanoparticles. Using a custom control program, set the function generator to produce a 10 cycle (3 &#181;s) HIFU burst, which is synchronized with the laser system. Also use this program to set a laser fluence of 0.4, 1.1, 2.1, or 3.4 mJ/cm2 though changing the timing between the triggering of the flash lamp firing and Q-switch opening in the laser system.</li>
	<li>Target the focal peak of the HIFU system 10 mm deep into the phantom, and at 13 unique locations, spaced by 5 mm, in the vertical direction. In each of these locations perform an exposure at a single peak negative HIFU pressure, with the four laser fluences stated in 4.2.</li>
	<li>Use the range of peak negative pressures 0, 0.91, 1.19, 1.43, 1.69, 1.92, 2.13, 2.34, 2.53, 2.71, 2.83, 3.00 and 3.19 MPa for the following exposure conditions: laser on in a nanoparticle free phantom, laser off in a nanoparticle phantom, and laser on in a nanoparticle phantom. To simulate a &#39;sham&#39; laser exposure, run the system as described, but shut the manual shutter on the output of the OPO. This approach will ensure that any RF noise generated will still be present to the PCD system.</li>
	<li>Program all settings and exposure positions into the control program, then execute to perform these measurements. PCD data is digitized and stored directly using the data acquisition card for post-processing. For each exposure parameter, 500 repeat exposures are acquired6.</li>
	<li>Process the broadband emissions detected by the PCD system from the short duration HIFU exposures into the phantoms using the technique detailed by McLaughlan et al. (2017)6.</li>
</ol>
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.
<ol>
	<li>Set the laser system to give a fluence of 3.4 mJ/cm2 and the function generator to give a CW exposure (every 330,000-cycle burst is synced to a laser pulse). In 11 unique locations in the phantom, select a peak negative pressure of 0.20, 0.62, 0.91, 1.19, 1.43, 1.69, 1.92, 2.13, 2.34, 2.53 or 2.71 MPa.</li>
	<li>Use a total exposure time of 17 s in order to acquire 1s of baseline before and after a 15 s CW HIFU exposure in the phantom. During this total exposure time, the data acquisition system is recording the PCD data. The microscope is connected to the control PC and the image frames are recorded during this time to provide a direct visualization of thermal lesion formation.</li>
	<li>Repeat the process in 4.3 for all the different exposure conditions outlined in 4.4.</li>
	<li>Process all PCD data off-line to calculate the inertial cavitation dose25 for each exposure.</li>
</ol>

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The author has nothing to disclose.

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&#177;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.

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 &#39;boiling&#39;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 &#39;contrast agents&#39; 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.

<table><tbody><tr><td>Single Element HIFU transducer</td><td>Sonic Concepts</td><td>H-102</td><td></td></tr><tr><td>55dB Power Amplifier</td><td>E&amp;amp;I</td><td>A300</td><td></td></tr><tr><td>Function Generator</td><td>Keysight Technologies</td><td>33250A</td><td></td></tr><tr><td>Differential Membrane Hydrophone</td><td>Precision Acoustics Ltd</td><td></td><td></td></tr><tr><td>TTL Pulse Generator</td><td>Quantum Composers</td><td>9524</td><td></td></tr><tr><td>Nd:YAG Pulse Laser</td><td>Continuum</td><td>Surelite I-10</td><td></td></tr><tr><td>OPO Plus</td><td>Continuum</td><td>Surelite</td><td></td></tr><tr><td>Fibre Bundle</td><td>Thorlabs Inc</td><td>BF20LSMA01</td><td></td></tr><tr><td>Energy Sensor</td><td>Thorlabs Inc</td><td>ES145C</td><td></td></tr><tr><td>Nanorods</td><td>Nanopartz</td><td>A12-40-850</td><td></td></tr><tr><td>Broadband detector</td><td>Sonic Concepts</td><td>Y-102</td><td></td></tr><tr><td>5 MHz high pass filter</td><td>Allen Avionics</td><td></td><td></td></tr><tr><td>40dB preamplifier</td><td>Spectrum GmbH</td><td>SPA.1411</td><td></td></tr><tr><td>14-bit data acquisition card</td><td>Spectrum GmbH</td><td>M4i.4420x8</td><td></td></tr><tr><td>Deionised Filtered Water</td><td>MilliQ</td><td></td><td></td></tr><tr><td>Acrylamide/Bis-acrylamide solution</td><td>Sigma Aldrich</td><td>A9927</td><td></td></tr><tr><td>1 mol/L TRIS Buffer</td><td>Sigma Aldrich</td><td>T2694</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma Aldrich</td><td>A3678</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>TEMED</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>3D printer</td><td>CEL-UK</td><td>Robox</td><td></td></tr><tr><td>3-axis positioning system</td><td>Zolix</td><td></td><td></td></tr><tr><td>Digital Microscope</td><td>Dino-lite</td><td>AM4113TL</td><td></td></tr><tr><td>Water Tank</td><td>Muji</td><td>Acrylic Tank</td><td></td></tr><tr><td>Optical Components</td><td>Thorlabs Inc</td><td>Various</td><td></td></tr><tr><td>Optomechanical Components</td><td>Thorlabs Inc</td><td>Various</td><td></td></tr><tr><td>BNC Cables</td><td>RS</td><td></td><td></td></tr><tr><td>Desktop PC</td><td>Custom Made</td><td></td><td></td></tr><tr><td>Hotplate Stirrer</td><td>Fisher</td><td></td><td></td></tr><tr><td>SBench6</td><td>Spectrum GmbH</td><td></td><td>Measurement software</td></tr></tbody></table>

controllable nucleation of cavitation from plasmonic gold nanoparticles for enhancing high intensity focused ultrasound applications

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 &#181;s), 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.

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 &#181;s 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58045/58045fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58045/58045fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58045/58045fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58045/58045fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58045/58045fig3.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58045/58045fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58045/58045fig4.jpg" /> 
Figure 4. Calculated inertial cavitation dose (ICD) recorded during exposures (a, b, e, &#38; f) with and (c &#38; d) without laser illumination. Peak negative pressure was either (a, c, &#38; e) 0.91 or (b, d, &#38; f) 2.53 MPa. The phantom used in (a &#38; b) did not contain any nanoparticles. <a href="https://www.jove.com/files/ftp_upload/58045/58045fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications at JoVE.com

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

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) ...

controllable-nucleation-cavitation-from-plasmonic-gold-nanoparticles

Nanyang Technological University

Research

JoVE Journal

Engineering

6.4K Views.  University of Leeds. 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.

Video: Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Chemistry

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Bioengineering

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to neutral gold Au(0) by reducing agents, such as sodium citrate or sodium borohydride. The extension of this method to decorate iron oxide or similar nanoparticles with gold nanoparticles to create multifunctional hybrid Fe2O3-Au nanoparticles is straightforward. This approach yields fairly good control over Au nanoparticle dimensions and loading onto Fe2O3. Additionally, the Au metal size, shape, and loading can easily be tuned by changing experimental parameters (e.g., reactant concentrations, reducing agents, surfactants, etc.). An advantage of this procedure is that the reaction can be done in air or water, and, in principle, is amenable to scaling up. The use of such optically tunable Fe2O3-Au nanoparticles for hyperthermia studies is an attractive option as it capitalizes on plasmonic heating of gold nanoparticles tuned to absorb light strongly in the VIS-NIR region. In addition to its plasmonic effects, nanoscale Au provides a unique surface for interesting chemistries and catalysis. The Fe2O3 material provides additional functionality due to its magnetic property. For example, an external magnetic field could be used to collect and recycle the hybrid Fe2O3-Au nanoparticles after a catalytic experiment, or alternatively, the magnetic Fe2O3 can be used for hyperthermia studies through magnetic heat induction. The photothermal experiment described in this report measures bulk temperature change and nanoparticle solution mass loss as functions of time using infrared thermocouples and a balance, respectively. The ease of sample preparation and the use of readily available equipment are distinct advantages of this technique. A caveat is that these photothermal measurements assess the bulk solution temperature and not the surface of the nanoparticle where the heat is transduced and the temperature is likely to be higher.

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

We describe the synthesis and properties of multifunctional Fe2O3-Au nanoparticles produced by a wet chemical approach and investigate their photothermal properties using laser irradiation. The composite Fe2O3-Au nanoparticles retain the properties of both materials, creating a multifunctional structure with excellent magnetic and plasmonic properties.

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

Cancer Research

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.