Institute of Fundamental Technological Research

Background:
The primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival.

Methods:
Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30 min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5 min. SonoVue® was injected intravenously (0.5 ml followed by 5 ml saline every 3.5 min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy.

Results:
The combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n = 10) tolerated an increased number of gemcitabine cycles compared with historical controls (n = 63 patients; average of 8.3 ± 6.0 cycles, versus 13.8 ± 5.6 cycles, p = 0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n = 10) was also increased from 8.9 months to 17.6 months (p = 0.011).

Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma.

Ultrasound, Microbubbles, Sonoporation, Pancreatic cancer, Image-guided therapy, Clinical trial

In research and industrial processes, it is increasingly common practice to combine multiple measurement modalities. Nevertheless, experimental tools that allow the co-linear combination of optical and ultrasonic transmission have rarely been reported. The aim of this study was to develop and characterise a water-matched ultrasound transducer architecture using standard components, with a central optical window larger than 10 mm in diameter allowing for optical transmission. The window can be used to place illumination or imaging apparatus such as light guides, miniature cameras, or microscope objectives, simplifying experimental setups.

Four design variations of a basic architecture were fabricated and characterised with the objective to assess whether the variations influence the acoustic output. The basic architecture consisted of a piezoelectric ring and a glass disc, with an aluminium casing. The designs differed in piezoelectric element dimensions: inner diameter, ID = 10 mm, outer diameter, OD = 25 mm, thickness, TH = 4 mm or ID = 20 mm, OD = 40 mm, TH = 5 mm; glass disc dimensions OD = 20–50 mm, TH = 2–4 mm; and details of assembly.

The transducers’ frequency responses were characterised using electrical impedance spectroscopy and pulse-echo measurements, the acoustic propagation pattern using acoustic pressure field scans, the acoustic power output using radiation force balance measurements, and the acoustic pressure using a needle hydrophone. Depending on the design and piezoelectric element dimensions, the resonance frequency was in the range 350–630 kHz, the −6 dB bandwidth was in the range 87–97%, acoustic output power exceeded 1 W, and acoustic pressure exceeded 1 MPa peak-to-peak.

3D stress simulations were performed to predict the isostatic pressure required to induce material failure and 4D acoustic simulations. The pressure simulations indicated that specific design variations could sustain isostatic pressures up to 4.8 MPa.The acoustic simulations were able to predict the behaviour of the fabricated devices. A total of 480 simulations, varying material dimensions (piezoelectric ring ID, glass disc diameter, glass thickness) and drive frequency indicated that the emitted acoustic profile varies nonlinearly with these parameters.

Ultrasound transducer, De-fouling, Optical window, Acoustic field simulation

Experimental research of ultrasound to induce or improve delivery has snowballed in the past decade. In our work, we investigate the use of low-intensity ultrasound in combination with clinically approved microbubbles to enhance the therapeutic efficacy of chemotherapy. Ten voluntary patients with locally advanced or metastatic pancreatic adenocarcinoma were consecutively recruited. Following standard chemotherapy protocol (intravenous infusion of gemcitabine over 30 min), a clinical ultrasoundscanner was targeted at the largest slice of the tumour using modified non-linear contrastimaging settings (1.9 MHz center frequency, 0.27 MPa peak-negative pressure), and SonoVue® was injected intravenously. Ultrasound and microbubble treatment duration was 31.5 min. The combined therapy did not induce any additional toxicity or increase side effect frequency when compared to chemotherapy alone. Combination treated patients were able to tolerate an increased amount treatment cycles when compare historical controls (n = 63); average of 8.3±6.0 cycles, versus 13.8±5.6 cycles. The median survival also increased from 7.0 months to 17.6 months (p = 0.0044). In addition, five patients showed a primary tumor diameter decrease. Combined treatment of ultrasound,microbubbles, and gemcitabine does not increase side effects and may have the potential to increase the therapeutic efficacy of chemotherapy in patients with pancreatic adenocarcinoma.

In this study, we analyse the behaviour of antibubbles when subjected to an ultrasonic pulse. Speci cally, we derive oscillating behaviour of acoustic antibubbles with a negligible outer shell, resulting in a Rayleigh Plesset equation of antibubble dynamics. Furthermore, we compare theoretical behaviour of antibubbles to behaviour of regular gas bubbles. We conclude that antibubbles and regular bubbles respond to an acoustic wave in a very similar manner if the antibubble's liquid core radius is less than half the antibubble radius. For larger cores, antibubbles demonstrate highly harmonic behaviour, which would make them suitable vehicles in ultrasonic imaging and ultrasound-guided drug delivery.

Studying the effects of ultrasound on biological cells requires extensive knowledge of both the physical ultrasound and cellular biology. Translating knowledge between these fields can be complicated and time consuming. With the vast range of ultrasonic equipment available, nearly every research group uses different or unique devices. Hence, recreating the experimental conditions and results may be expensive or difficult. For this reason, we have developed devices to combat the common problems seen in state-of-the-art biomedical ultrasound research. In this paper, we present the design, fabrication, and characterisation of an open-source device that is easy to manufacture, allows for parallel sample sonication, and is highly reproducible, with complete acoustic calibration. This device is designed to act as a template for sample sonication experiments. We demonstrate the fabrication technique for devices designed to sonicate 24-well plates and OptiCell™ using three-dimensional (3D) printing and low-cost consumables. We increased the pressure output by electrical impedance matching of the transducers using transmission line transformers, resulting in an increase by a factor of 3.15. The devices cost approximately €220 in consumables, with a major portion attributed to the 3D printing, and can be fabricated in approximately 8 working hours. Our results show that, if our protocol is followed, the mean acoustic output between devices has a variance of <1%. We openly provide the 3D files and operation software allowing any laboratory to fabricate and use these devices at minimal cost and without substantial prior know-how.

Sonoporation, experimentation devices, rapid prototyping, ultrasound transducers

An antibubble consists of a liquid droplet, surrounded by a gas, often with an encapsulating shell. Antibubbles of microscopic sizes suspended in fluids are acoustically active in the ultrasonic range. In this study, a Rayleigh-Plesset-like model is derived for micron-sized antibubbles encapsulated by Newtonian fluids. The theoretical behaviour of an encapsulated antibubble is compared to that of an antibubble without an encapsulating shell, a free gas bubble, and an encapsulated gas bubble. Antibubbles, with droplet core sizes in the range of 60– 90% of the equilibrium antibubble inner radius were studied. Acoustic pressures of 100kPa and 300kPa were studied. The antibubble resonance frequency, the phase difference of the radial oscillations with respect to the incident acoustic pulse, and the presence of higher harmonics are strongly dependent of the core droplet size. The contribution to the radial dynamics from a zero-thickness shell is negligible for the bubble size studied, at high acoustic amplitudes, antibubbles oscillate highly nonlinearly independent of core droplet size. This may allow for active leakage detection using harmonic imaging methods.

Active leakage detection, Antibubble, Bubble resonance, Microbubble, Nonlinear dynamics

Purpose
Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50 % are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer.

Experimental Design
An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound.

Results
Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10 % demonstrated in comparison to the control groups.

Conclusion
Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation.

Sonoporation, Pancreatic cancer, Ultrasound, Chemotherapy, 3D ultrasound, Bioluminescence

Purpose:
The purpose of this study was to investigate the ability and efficacy of inducing sonoporation in a clinical setting, using commercially available technology, to increase the patients’ quality of life and extend the low Eastern Cooperative Oncology Group performance grade; as a result increasing the overall survival in patients with pancreatic adenocarcinoma.

Methods:
Patients were treated using a customized configuration of a commercial clinical ultrasound scanner over a time period of 31.5 min following standard chemotherapy treatment with gemcitabine. SonoVue® ultrasound contrast agent was injected intravascularly during the treatment with the aim to induce sonoporation.

Results:
Using the authors’ custom acoustic settings, the authors’ patients were able to undergo an increased number of treatment cycles; from an average of 9 cycles, to an average of 16 cycles when comparing to a historical control group of 80 patients. In two out of five patients treated, the maximum tumor diameter was temporally decreased to 80 ± 5% and permanently to 70 ± 5% of their original size, while the other patients showed reduced growth. The authors also explain and characterize the settings and acoustic output obtained from a commercial clinical scanner used for combined ultrasound microbubble and chemotherapy treatment.

Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available clinical ultrasound scanners to increase the number of treatment cycles, prolonging the quality of life in patients with pancreatic adenocarcinoma compared to chemotherapy alone.

Ultrasound, Microbubbles, Sonoporation, Chemotherapy

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Ultrasound, Microbubbles, Physical gene delivery method, Gene therapy

Electron beam lithography (EBL) was used to fabricate microchannels to produce microbubbles with highly homogeneous size distributions. Using the EBL technique, microchannels can be prototyped at a fast and cost effective rate allowing for evaluation of various microbubble shell materials.

Microbubbles, EBL, Targeted drug delivery

Introduction
High-intensity focused ultrasound is finding increasing therapeutic use. However, the frequencies at which it operates are typically limited to below 5 MHz, preventing research into therapy with ultrahigh spatial precision. A reason for this is that the design and fabrication of high-frequency biomedical ultrasound transducers to produce high intensities is an engineering challenge, especially for operating frequencies above 30 MHz, primarily because of the acoustic impedance mismatch and the high attenuation of water of 6dB/cm at 50 MHz leading to a low penetration depth. Commonly used materials such as PZT do not have the ability to produce a high enough intensity, due to de-poling or cracking. A potential application of high-intensity high-frequency ultrasound is non-invasive microsurgery.

Methods
To overcome these problems, we used Y-36o Lithium Niobate (LiNbO3). This crystal has a high Curie temperature and is much more difficult to de-pole at high-power inputs. In addition, Y-36o LiNbO3 has a resonant frequency of 3.3 MHz mm-1, thus allowing for much thicker elements at higher frequencies compared to PZT. A bowl transducer was manufactured using a total of 7 0.5-mm thick elements (4 hexagonal and 5 pentagonal) with a maximum width of 25 mm. The bowl had a curvature radius of 50 mm. The transducer was microballoon-backed in order to simplify the manufacturing process. The pentagonal elements were linked and driven by a 50-dB amplifier, whereas the hexagonal elements were linked and driven by a 55-dB amplifier. To test the available working frequency; single element transducers were manufactured with element thickness ranging from 500 μm to 200 μm, having working frequencies between 6.6 MHz and 20 MHz.

Results
The multi-element focused transducer generated a modulated sound field with an enveloped wavelength of 550 kHz at a frequency of 6.6 MHz with a maximum peak-to-peak pressure of 24.3 MPa; equivalent to mechanical index of 4.7. The modulation could be varied by changing the phase of either the pentagonal or hexagonal linked elements. The microballoon-backed transducers had a 5% reduced acoustic output compared to the air-backed transducer. Single- element transducers produced a maximum peak-to-peak pressure of 14 MPa at 6.3 MHz in the acoustic focus at 12 mm. These transducers were capable of producing over 6 MPa and 4 MPa at the 3rd and 5th harmonics, respectively, corresponding to frequencies of 21 MHz and 35 MHz.

Conclusion
We have established that manufacturing a high frequency, high intensity, multi-element, focused ultrasound transducer using LiNbO3 is feasible. We have also shown it is possible to use the resonant frequency and up to the 5th harmonic to achieve higher working frequencies.

High-frequency ultrasound, Ultrasound transducer, MR-guided Focussed Ultrasound Surgery

Focused ultrasound surgery (FUS) is usually based on frequencies below 5 MHz—typically around 1 MHz. Although this allows good penetration into tissue, it limits the minimum lesion dimensions that can be achieved. In this study, we investigate devices to allow FUS at much higher frequencies, in principle, reducing the minimum lesion dimensions. Furthermore, FUS can produce deep-sub-millimeter demarcation between viable and necrosed tissue; high-frequency devices may allow this to be exploited in super cial applications which may include dermatology, ophthalmology, treatment of the vascular system, and treatment of early dysplasia in epithelial tissue. In this paper, we explain the methodology we have used to build high-frequency high-intensity transducers using Y-36°-cut lithium niobate. This material was chosen because its low losses give it the potential to allow very-high- frequency operation at harmonics of the fundamental operating frequency. A range of single-element transducers with center frequencies between 6.6 and 20.0 MHz were built and the transducers’ e ciency and acoustic power output were measured. A focused 6.6-MHz transducer was built with multiple elements operating together and tested using an ultrasound phantom and MRI scans. It was shown to increase phantom temperature by 32°C in a localized area of 2.5 × 3.4 mm in the plane of the MRI scan. Ex vivo tests on poultry tissue were also performed and shown to create lesions of similar dimensions. This study, therefore, demonstrates that it is feasible to produce high-frequency transducers capable of high-resolution FUS using lithium niobate.

Lithium Niobite, Ultrasound Transducer, MRI-Guided ultrasound, Microsurgery

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5 × 10^5 BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Gene delivery, Sonoporation, Tendon

Acoustic cavitation can occur in therapeutic applications of high-amplitude focused ultrasound. Studying acoustic cavitation has been challenging, because the onset of nucleation is unpredictable. We hypothesized that acoustic cavitation can be forced to occur at a specific location using a laser to nucleate a microcavity in a pre-established ultrasound field. In this paper we describe a scientific instrument that is dedicated to this outcome, combining a focused ultrasound transducer with a pulsed laser. We present high-speed photographic observations of laser-induced cavitation and laser- nucleated acoustic cavitation, at frame rates of 0.5×106 frames per second, from laser pulses of energy above and below the optical breakdown threshold, respectively. Acoustic recordings demonstrated inertial cavitation can be controllably introduced to the ultrasound focus. This technique will contribute to the understanding of cavitation evolution in focused ultrasound including for potential therapeutic applications.

Laser-nucleated acoustic cavitation, focussed ultrasound

Introduction
Focussed ultrasound surgery can heat tissue to a temperature that causes protein denaturation and coagulative necrosis. For high-resolution focussed ultrasound microsurgery, high working frequencies are necessary. The manufacture of such equipment is technically challenging. Low acoustic amplitudes have been associated with increased cellular drug and gene uptake.
We studied tissue, microbubbles and cells, using commercial and self-built transducers operating at various frequencies, using very low or very high acoustic amplitudes.

Methods
We manufactured a high-frequency, high-intensity focussed ultrasound transducer, using lithium niobate as the active element.
To study acoustic cavitation, we designed and built a scientific instrument combining a pulsed laser and a high-intensity focussed ultrasound transducer, capable of nucleating at precise locations. The cavitation dynamics were recorded using high-speed cameras. At high acoustic intensities, interacting cavitation clouds were formed.

Results
Clusters formed at a quarter wavelength apart owing to radiation forces. We observed cluster coalescence and translation towards the capillary wall.
Microbubbles under sonication have been observed to create transient pores in adjacent cell membranes, also known as sonoporation. We observed lipid-shelled microbubbles near cancer cells under quasi-continuous low-amplitude sonication. Typically within a second of sonication, microbubbles were seen to enter the cells and dissolve. This new explanation of sonoporation was verified using high-speed photography and confocal fluorescence microscopy.
Our custom-built transducer was capable of creating 2.5×3.4 (mm)2 lesions without affecting surrounding tissue.
Such disruptive effects of ultrasound also have applications outside medicine. Since cyanobacteria contain gas vesicles, we hypothesised that these can be disrupted with the aid of ultrasound. During 1-hour sonication in the clinical diagnostic range, we forced blue-green algae to sink, thus promoting natural decay.

Conclusion
If drug and genes can be successfully coupled to acoustically active vehicles, sonoporation might revolutionise noninvasive therapy as we know it.

Therapeutic ultrasound, Sonoporation

The purpose of this study was to investigate the physical mechanisms of sonoporation, in order to understand and improve ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation and resealing of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. The authors studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6?6 MHz ultrasound with a mechanical index of 0?2 and observed with a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell while entering. The authors have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic agent is added to the shell of the bubble or inside the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. In addition, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Sonoporation, Low mechanical index, Microbubbles, Ultrasound contrast agent, HeLa cells, Cell penetration

The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source.
In this paper, we analyse how ultrasound contrast agent clusters are formed in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1– 10 MHz. Furthermore, we show high-speed camera footage of microbubble clustering phenomena.
We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces.
We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. It also just takes seconds to drive the clusters towards the capillary wall.
Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation.

Capillary blocking, Embolisation, Microfoam, Radiation forces, Ultrasound contrast agent

Algae are aquatic organisms classified separately from plants. They are known to cause many hazards to humans and the environment. Algae strands contain nitrogen-producing cells that help them float (heterocysts). It is hypothesized that if the membranes of these cells are disrupted by means of ultrasound, the gas may be released analogous to sonic cracking, causing the strands to sink. This is a desirable ecological effect, because of the resulting suppressed release of toxins into the water.
We subjected small quantities of blue-green algae of the Anabaena sphaerica species to ultrasound of frequencies and pressures in the clinical diagnostic range, and observed the changes in brightness of these solutions over time. Blue-green algae were forced to sink at any ultrasonic frequency we studied, supporting our hypothesis that heterocysts release nitrogen under ultrasound insonification in the clinical diagnostic range.
Although the acoustic fields we used to eradicate blue-green algae are perfectly safe in terms of mechanical index, the acoustic pressures surpass the NURC Rules and Procedures by over 35 dB. Therefore, caution should be taken when using these techniques in a surrounding where aquatic or semi-aquatic animals are present.

Ultrasonic algae eradication, Blue-green algae, Sonic cracking

An antibubble consists of a liquid droplet, surrounded by a gas, often with an encapsulating shell. Antibubbles of microscopic sizes suspended in fluids are acoustically active in the ultrasonic range. Antibubbles have applications in food processing and guided drug delivery. We study the sound generated from antibubbles, with droplet core sizes in the range of 0–90% of the equilibrium antibubble inner radius. The antibubble resonance frequency, the phase difference of the echo with respect to the incident acoustic pulse, and the presence of higher harmonics are strongly dependent of the core droplet size. Antibubbles oscillate highly nonlinearly around resonance size. This may allow for using antibubbles in clinical diagnostic imaging and targeted drug delivery.

Antibubble, Nonlinear, Echo, Rayleigh-Plesset, Targeted drug delivery

Hydroelectric power is a clean source of energy, providing up to 20% of the World's electricity. Nevertheless, hydroelectric power plants are plagued with a common problem: silt. The silt causes damage to turbine blades, which then require repairing or replacing. In this study, we investigated the possibility to filter micron-sized particles from water using ultrasound. We designed a custom-made flow chamber and performed flow simulations and experiments to evaluate its efficacy. We used a 195-kHz ultrasound transducer operating in continuous-wave mode with acoustic output powers up to 12W. Our acoustic simulations showed that it should be possible to force a 200-μm particle over 2cm in flow, using an acoustic pressure of 12 MPa. Our flow simulations showed, that the fluid flow is not drastically decreased with the flow chamber, which was validated by the experimental measurements. The flow was not reduced when the ultrasound was activated. The acoustic filtering was effective between acoustic powers of 2.6 and 6.4W, where the particle concentration in the clean output was statistically significantly lower than the null experiments.

Acoustic filtering

Therapeutic ultrasound has been in use for over 70 years but has primarily been a thermal modality. Sonoporation, the use of ultrasound and stable gas microbubbles in the size range of 2-10 μm to form transient pores in cell membranes, has been of great interest in the past 15 years. This technique could be used to improve the delivery of current drugs in very localised regions. There are several phenomena behind sonoporation that all occur non-exclusively: push, pull, jetting, inertial cavitation, shear and, translation. Pre-clinical work has shown that sonoporation can be used to reduce primary tumour burden and inhibit metastatic development. Our clinical trial showed that ultrasound in combination with microbubbles and chemotherapy can effectively double the number of chemotherapy cycles patients can undergo, meaning that the patients were healthier for a longer period of time. Nevertheless, sonoporation is still in its infancy and there is vast room for improvement in both the areas of microbubbles and ultrasound.

Sonoporation

Clinical diagnostic ultrasound has been known as one of the safest imaging modalities available, yet very little is known about the cellular response to such acoustic conditions. With the increased interest in therapeutic ultrasound it is becoming ever more important to understand the effects of ultrasound on cells.In our work here we investigate the effect of clinical diagnostic ultrasound on several cell signalling proteins (p38 p-Thr180/p-Tyr182, ERK 1/2 p-Thr202/p-Tyr204 and p53 ac-Lys382) on leukaemia cells (MOLM-13) and monocytes. Our results show that leukaemia cells and monocytes react differently to ultrasound and microbubbles. A relatively small increase in p38 signalling was seen in the leukemic cells, and only at higher intensities in combination with microbubbles. In contrast the monocytes showed an increase in p38 signalling at all acoustic intensities with microbubbles and at the high acoustic intensity without microbubbles. Furthermore, the leukemic cells showed an overall increase in ERK 1/2 signalling whereas the monocytes showed a decrease. These results indicate that the leukaemia cells are less sensitive to stress induced by ultrasound and microbubbles when compared to normal monocytes. In conclusion, our results show that clinical diagnostic ultrasound does have a measurable effect on intracellular signalling but may differ drastically between different cell types. This may affect the conditions necessary for therapeutic ultrasound.

Phospho-signaling pathways, Ultrasound contrast agent

Glass windowed ultrasound transducers have several potential uses ranging from multi-modal research (ultrasound and optics) to industrial application in oil and gas or chemistry. In our work here we compare four different designs for transparent glass windowed ultrasound transducers. Each design was characterised using field scanning, radiation force measurements, frequency sensitivity measurement and FEM simulations. Field scans showed that small variations in design can greatly affect the size and location of the acoustic focus. The results coincided with those seen in the simulations. Radiation force measurements showed that the devices were able to easily exceed acoustic powers of 10W, with efficiencies of up to 40%. Isostatic simulations shows that the design also affects the physical strength of the devices. Current designs were able to withstand between 300 and 700 psi on the front surface. The devices were cost effective due to the minimal amount of materials necessary and the simple fabrication process. More work needs to be done to improve the power output and stress handling capabilities.

Ultrasound transducer

Separating oil, gas, and water is a slow, and therefore expensive, process, especially if the gas bubbles and oil droplets are very small. Yet, with increasingly strict regulations on filtered sea water quality, it is a process of major importance to most oil and gas industry. Using customised ultrasonic devices, we have been able to drive microbubbles through saturated fluids, forcing the bubbles to cluster and form microfoams at equal distances. These microfoams were then driven out of the fluid. In this presentation highspeed photography footage of this process will be shown. Ultrasound-assisted separation is a cheap technique that may have applications on a much bigger scale.

Ultrasound, Oil, gas and water separation

The purpose of this study was to investigate the physical mechanisms of sonoporation, to understand and ameliorate ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation of a cell membrane with help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. We studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6.6-MHz ultrasound with a mechanical index of 0.2 and observed with a high- speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell whilst entering. We have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic load is added to the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. However, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Sonoporation, Ultrasound, Microbubbles, HeLa Cancer cells, Drug delivery, Low Mechanical Index

Ultrasound as a therapeutic means is increasing in popularity, owing to its non-invasiveness and low cost. Biomedical applications include surgery, cyanobacteria eradication, and drug and gene delivery. Recent advances have been associated with the ultrasound-induced formation of transient porosities in cell membranes. These and other applications have been associated with the behaviour of medical microbubbles near cells.
Common methods of studying the dynamic behaviour of microbubbles are high-speed photography and optical microscopy.
Here, we give a brief overview of our fields of study in biomedical ultrasonics, concentrating on bubble–cell interactions.
There are three standards for the safe use of biomedical sound. When introducing bubbles, these safety guidelines may be misleading.

Ultrasound, Sonoporation, Safety guidelines

Focused ultrasound surgery (FUS) is usually based on frequencies below 5 MHz, typically around 1 MHz. Whilst this allows good penetration into tissue, it limits the minimum lesion dimensions that can be achieved. In the study reported here, we investigated devices to allow FUS at much higher frequencies, therefore in principle reducing the minimum lesion dimensions. We explain the methodology we have used to build high-frequency high-intensity transducers using Y-36o cut lithium niobate. This material was chosen as its low losses give it the potential to allow very high-frequency operation at harmonics of the fundamental operating frequency. A range of single element transducers with a centre frequency between 6.6 MHz and 20.0 MHz was built and the transducers’ efficiency and acoustic power output were measured. A focussed 6.6-MHz transducer was built with multiple elements operated together and tested using an ultrasound phantom and MRI scans. It was shown to increase phantom temperature by 32OC in a localised area of 2.5 mm × 3.4 mm in the plane of the MRI scan. This study therefore demonstrates that it is feasible to produce high-frequency transducers capable of high-resolution focused ultrasound surgery using lithium niobate.

FUS, high frequency, lithium niobate, high resolution, transducer manufacture, MRI compatibility

The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source. Alternatively, the microfoam could be removed from the blood pool. The latter technique might be applicable in highly toxic ultrasound-guided drug delivery. We analysed how ultrasound contrast agents with different shell compositions form clusters in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1–10 MHz. We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces.
We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. The clusters contain approximately 2,000 ultrasound contrast agent microbubbles. Clusters streaming trough a capillary interact, forming morphing microfoam. Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation, including the release of its contents.

Algae have been proven to be a severe health hazard to humans, aquatic and semi-aquatic animals. Chemical methods available to control the algae have unwanted side-effects. For this reason, ultrasonic algae control has been under investigation. We measured the eradication effectiveness of ultrasound at three typical centre frequencies. At all three frequencies physical damage to the algae was observed. We conclude that it is possible to eradicate blue-green algae in the clinical diagnostic range. Taking into account the geometry, the low attenuation in water, and the NATO Undersea Research Centre for Human Diver and Marine Mammal Risk Mitigation Rules and Procedures, even at these low voltages, the safe swimming distance is at least several meters away from the sound source.

Ultrasound, Algae eradication

We study the sonication of stable particles that encapsulate a liquid core, so-called antibubbles. Acoustically active antibubbles can potentially be used for ultrasound-guided drug delivery. In this presentation, we derive the oscillating behaviour of acoustic antibubbles with a negligible outer shell, resulting in a Rayleigh-Plesset equation of antibubble dynamics. Furthermore, we compare the theoretical behaviour of antibubbles to that of regular gas bubbles. We conclude that antibubbles and regular bubbles are acoustically active in a very similar way, if the liquid core is less than half the antibubble radius. For larger cores, antibubbles demonstrate highly harmonic behaviour, which would make them suitable vehicles in ultrasonic imaging and ultrasound-guided drug delivery.

Harmonics, Antibubbles

Background: Pancreatic Adenocarcinoma (PDAC) represents one of the most lethal human cancers. Surgery is often unfeasible, and the tumors respond poorly to radiation or chemotherapeutic drugs. Consequently, pancreatic cancer represents a huge burden to society and the need for new therapeutic options is evident. Experimental research using ultrasound to improve therapeutic delivery has soared in the past decade. We aimed toevaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary goal was to develop a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance grade, and prolongation of survival. Methods: Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30 min. Subsequently patients were treated using a commercial clinical ultrasound scanner for 31.5 min. SonoVue was injected intravenously (0.5 ml followed by 5 ml saline every 3.5 min) during the ultrasound treatment with the aim of enhancing therapeutic efficacy. Results: The combined therapeutic regimen did not induce any additional toxicity or increase side effect frequency when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n = 10) tolerated an increased number of gemcitabine cycles compared with historical controls (n = 63 patients; average of 8.3±6.0 cycles, versus 13.8±5.6 cycles). In five patients, the maximum tumor diameter was decreased during treatment. The median survival in our patients was also increased from 7.0 months to 17.6 months (p = 0.0044). Conclusions: We perform the first-in-human study evaluating the toxicity and efficacy of ultrasound and microbubble enhanced chemotherapy. It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting with no additional toxicity. This combined treatment may improve the clinical efficacy of chemotherapeutic agents, prolong the quality of life, and extend survival in patients with PDAC. Clinical trial information: NCT01674556.

Bubbles are emptiness, microscopic clouds surrounded by the world. Born by chance with a violent and brief life, collapsing in a union with the infinite [1]. Remarkably similar to the life of a human being. However, there are some discrepancies when comparing the life of a bubble, and that of a human being. For example, the nature of the acoustically active bubble is described by the language of mathematics, where understanding the behaviour of bubble dynamics has been a goal for physicists for centuries [2]. However, not only physicists have been enchanted by the intrinsic beauty encompassed in the simplicity of a bubble. Throughout history bubbles have been a symbol in art.

Antibubbles