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Transcranial ultrasound stimulation offers a non-invasive and targeted option for neuromodulation of the brain. However, the clinical utility of transcranial ultrasound for brain function has been impeded by the highly variable attenuation and phase distortions of the human skull. A method to focus ultrasound using a target-specific acoustic lens at the temporal bone window is presented. Ultrasound frequency (500 kHz), transducer aperture size (d = 64 mm), and lens material (PDMS) were selected based on acoustic simulations (k-Wave). Through computational modeling, the utility of this concept was confirmed for deep brain targets such as the perirhinal cortex, anterior hippocampus, globus pallidus internus, and subthalamic nucleus. Experimental validation of the work on nine target-specific lenses for use through two cadaver skull samples confirmed reliable predictions by the acoustic model. To evaluate this method for clinical application for neuromodulation of Parkinson's disease, interference between ultrasound waveforms and implanted electrodes was studied. Two ways were found to best mitigate the observed mutual interference: (i) forming the acoustic focus away from the electrode tip and (ii) using ultrasound pulse repetition frequencies higher than those of brain signal bands. Taken together, this method of case-specific stimulation delivery with an acoustic lens guides the design and workflow of ultrasound-mediated deep brain stimulation at the temporal bone window for effective, safe, and reproducible transcranial ultrasound stimulation. Results from this project are being implemented in an upcoming clinical trial to demonstrate its efficacy.

Gene delivery will cure all. That was the hope of many since the discovery and therapeutic implementation of genetic research. Although some of that hope has been realised, the delivery of nucleotides to cells in the body remains a major barrier in wider application. Ultrasound-mediated delivery of drugs has been investigated in various forms for decades and continues to be an area of engineering and science that is much explored. The body of work presented in this thesis strides to bring the two areas of research endeavour together. Ultrasound-mediated delivery of drugs and antibody particles, normally in conjunction with cavitation nucleation agents, has been shown to be effective both in vitro and in vivo. In this work, we focus on nucleotide delivery, in particular mRNA, which has yet to be fully explored, especially in the context of achieving delivery of non-encapsulated, 'free' mRNA. Data presented here explores the relationship between cavitation and the transfer of, and protein expression from, free mRNA. The protein expression exhibited in various cell lines in vitro was further tested in vivo, using either sub-micron or micron-sized cavitation nuclei and the effects on transfection efficiency and expression time were examined. A luciferase reporter gene assay was used to characterise events in vitro across different cell lines including cancer and non-cancerous types. The impact and varying acoustic exposure parameters and cavitation on expression were assessed, and differences in expression time and transfection rate were observed. These data were used to determine appropriate acoustic parameters for in vivo delivery. Cavitation-mediated delivery was examined in vivo in two types of cancer tumours, and mRNA expression was quantified using in vivo fluorescence imaging and using excised homogenised tumour tissue. The results presented here lay the foundations for cavitation-mediated delivery and transfection of free mRNA both in vitro and in vivo by identifying appropriate acoustic parameters and cavitation nucleation agents, which result in enhanced delivery and by identifying the appropriate time windows for measurements to quantify expression.

The use of High Intensity Focussed Ultrasound for the thermal ablation of renal tumours provides a completely non-invasive method for treatment, which is advantageous for patients who are not eligible for surgery due to co-morbidity issues. However, clinical trials have shown variable results with challenges to the delivery of the ultrasound beam related to the intervening tissue layers. The use of patient-specific models is investigated as a means to provide a treatment planning step to aid clinicians in the choice of the transducer's position and ultrasound power for the treatment. CT scans from patients treated in a clinical trial were segmented into bone, muscle and fat. Then patient-specific acoustic simulations were undertaken using the time domain acoustic solver software k-Wave. Patient-to-patient anatomical variability yielded differences of up to 154 W in the acoustic powers required for successful renal tumour ablation. To find an optimal placement for the transducer, back-propagation acoustic simulations were undertaken. A virtual source was placed within the tumour with the receiver surface representing the potential transducer locations. The complex pressure received over the transducer surface was used to predict the optimal placement. The present thesis also considers the sensitivity of the model to speed of sound variations. There was a significant impact on the focal pressure due to the relative change in speed of sound between fat and muscle. For the patient model tested there was a > 20% change in maximum pressure for variations of 4% and 7% in the speed of sound of muscle and fat respectively. In conclusion, patient-specific acoustic modelling was successfully used to evidence the patient to patient variability in power requirements for renal tumour ablation as well as the significance of transducer placement for the individual patient. This in turn explains the significant variability in ablation outcomes achieved in clinical studies to date, and emphasizes the need for careful patient-specific treatment planning if renal ablation is to be adopted clinically.

Non-invasive brain stimulation techniques allow for excitation or inhibition of neural activity via externally applied stimuli. Recently, ultrasound (US) has been shown to stimulate neurons with better spatial localisation than existing electrical stimulation modalities. We present a review of the literature in both the central and peripheral nervous systems demonstrating a range of effects from de novo action potential generation in isolated cell preparations to cognitive responses in humans. However, several questions remain before the technique can become widespread. These include: methods to accurately model, and deliver, transcranial US to specific brain targets; the safety of the approach from both a mechanical and thermal standpoint; elucidation of the fundamental mechanisms through which acoustic perturbations result in nervous system modulation; and the development of monitoring techniques to confirm US focusing in vivo as well as to detect the induced effects. In this thesis, we focus on the delivery and safety aspects. We present numerical simulations using k-Wave and experimental measurements demonstrating focusing to areas of the visual cortex using ex-vivo human skulls with single-element transducers. We show that targeting of many areas can be achieved without the use of lenses or arrays. Furthermore, we conduct an in-depth safety review detailing the parameter spaces over which studies have been conducted and highlighting any damage areas. In current human studies the technique appears to be safe with minimal, if any, side effects. Finally, we report findings from a human pilot study with the aim of modulating sites within the visual cortex. We present direct evidence of strong non-specific auditory activation following bursts of US confirming previous animal studies. These effects can confound US neuromodulation trials and demands further attention before US can realise its potential as both a scientific instrument to probe brain function and as a therapeutic modality to modulate brain activity.

A major challenge in chemotherapy is that the systemic delivery of drugs to both healthy tissue as well as the tumour results in unwanted side effects. Thermosensitive liposomal carriers are designed to release their payload when they are exposed to mild hyperthermia (a few degrees above body temperature). If a tumour can be locally heated, thermosensitive liposomes can provide triggered and localised delivery of the drug which should reduce systemic toxicity. Ultrasound is a noninvasive modality which can be used to produce localised heating at depth. However, most clinically approved high-intensity focused ultrasound (HIFU) systems are designed for tumour ablation and exhibit a focal volume which typically spans 1-3mm in width and 10-15mm in length, generating temperatures in excess of 50◦C for a few seconds to directly kill tissue within the focal volume. Hyperthermia-triggered drug release usually requires maintaining a tumour volume which often spans several centimetres in size at a consistent therapeutic temperature of 39.5-42◦C for durations up to an hour. This is difficult to achieve for most current HIFU transducers due to their small focal volume. The approach employed here is to design an acoustic lens that retrofits onto a clinical HIFU system to produce a focal volume better suited for mild hyperthermia. 3D printing is employed in this work to produce the lenses, and sectored lenses which enable a single-element transducer to produce acoustic fields with multiple foci in an annular pattern are investigated. Experimental measurements of the pressure and temperature using a small therapeutic transducer are consistent with simulations, and the simulated -3dB free-field focal volume of the transducer was increased from 9.6mm3 to 90.9mm3. Finite element software is used to perform 3D linear full-wave simulations of an acoustic lens in a human liver target derived from a segmented CT dataset. The computational challenges with performing large 3D finite element simulations are addressed by dividing the simulation domain into several subregions. A phase-conjugate lens combined with masking transducer locations associated with ribs is explored as a solution to address aberrations in the acoustic field caused by tissue inhomogeneity. Experiments with a sectored acoustic lens on a clinical HIFU system showed the radial location of the free-field focus was shifted from 0mm to ±2.8mm in an annular pattern. This work shows that retrofitting a HIFU system with an acoustic lens is a practical approach to broadening the focal spot.

Shock wave lithotripsy is a non-invasive procedure by which kidney stones are fragmented. Currently, many shock waves are delivered to the body that do not impact the stone, but do result in tissue trauma, mainly caused by cavitation. This work presents a monitoring system to reduce the number of shock waves delivered to the body, by tracking the target, and to detect cavitation throughout the procedure. For the first goal, a circular array, housing twenty-two 0.5 MHz transducers that can be mounted around the aperture of a clinical lithotripter, was used to collect pulse-echo data, from which an acoustic triangulation algorithm was able to locate stones. The accuracy of the system in water was better than 4 mm in the lateral direction and better than 2 mm in the axial direction within ±15 mm of the focus-which is the variation that would be expected clinically. For the second goal, the acoustic pressure field of a shock wave therapy source was measured and its cavitation field was determined. Cavitation was localised using a conventional ultrasound probe and passive acoustic mapping. Bubbles were shown to grow, collapse, and rebound, and the characteristic times were determined. It was found that these parameters increased with the energy level and the pulse repetition frequency and that at the highest energy setting the characteristic time was 288 μs. Finally, the characteristic time of cavitation in an ex vivo pig kidney and for an artificial stone in water due to a clinical shock wave source was determined using passive acoustic mapping. The characteristic times in the pig kidney were shorter than for the stone, e.g. 560 μs in the kidney and 730 μs on the stone at EL 6. No significant correlation between cavitation on a stone that fractured well and a stone that fractured poorly could be determined. The work in this thesis heralds an advance in current lithotripsy monitoring techniques that are based on B-mode and x-ray. Real-time tracking of the kidney stone and cavitation mapping throughout the treatment would limit the renal injury that routinely occurs during lithotripsy.

Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inflammatory responses. However, they are challenged by limited delivery. Thus, more efficient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they offer a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection. This work presents the first investigation on the efficacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that defines the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically significant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to effect gene inhibition through the delivery of siRNA. Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was significantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal. The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation effects and isolate effects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery. A final report was on the significant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus - focused ultrasound (US), direct comparisons were made between SWs and US on the efficiency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.

This thesis presents a combined numerical and experimental study on the response of kidney cells to shock waves. The motivation was to develop a mechanistic model of cell deformation in order to improve the clinical use of shock waves, by either enhancing their therapeutic action against target cells or minimising their impact on healthy cells. An ultra-high speed camera was used to visualise individual cells, embedded in tissue-mimicking gel, in order to measure their deformation when subject to a shock wave from a clinical shock wave source. Advanced image processing was employed to extract the contour of the cell from the images. The evolution of the observed cell contour revealed a relatively small deformation during the compressional phase and a much larger deformation during the tensile phases of a shock wave. The experimental observations were captured by a numerical model which describes the volumetric cell response with a bilinear Equation of State and the deviatoric cell response with a viscoelastic framework. Experiments using human kidney cancer cells (CAKI-2) and noncancerous kidney cells (HRE and HK-2) were compared to the model in order to determine their mechanical properties. The differences between cancerous and noncancerous cells were exploited to demonstrate a design process by which shock waves may be able to improve the specificity on targeted cancer cells while having minimal effect on normal cells. The cell response to shock waves was studied in a more biophysically realistic environment to include influence of cell size, shape and orientation, and the presence of neighbouring cells. The most significant difference was predicted when cells were in a cluster in which case the presence of neighbouring cells resulted in a four-fold increase on the von Mises stress and the membrane strain. Finally the numerical model was extended to capture the effect of cell damage using one of two paradigms. In the first paradigm the model captured microdamage during one shock wave but then assumed that the cell recovered by the time the next shock wave arrived. The second model allowed microdamage to accumulate with increasing number of shock waves. These models may be able to explain the strong effect that shock wave loading rate has on tissue damage. In conclusion a validated numerical model has been developed which provides a mechanistic understanding of how cells respond to shock waves. The model has application in suggesting improved strategies for current uses of shock waves, e.g., lithotripsy, as well as opening up new indications such as cancer treatment.

High-intensity focused ultrasound (HIFU) therapy is a non-invasive therapy method for the treatment of solid tumours. One of the main barriers to wider clinical adoption of HIFU is the lack of reliable and cost-efficient therapy monitoring. One promising technique is acoustic radiation force (ARF) ultrasound elastography, which relies on the temperature dependence of tissue properties to affect displacement amplitudes. An extensive literature review of tissue acoustic, thermal and mechanical properties identified an absence of data on the temperature dependence of the viscoelastic properties. Experiments were carried out in ex vivo liver to determine these properties over the range of temperatures and frequencies relevant to ARF. ARF induced displacements were quantified experimentally in a tissue-mimicking phantom. The comparison of sine and square modulated ARF excitation showed that square modulation results in higher peak amplitudes and smaller second harmonic components in the frequency domain. A simulation model was developed, which accurately reproduced the observed displacements with different modulation frequencies and power levels. The effect of temperature on ARF displacements was measured experimentally during HIFU therapy in ex vivo liver. Simulations captured the same phenomena as observed in the experiments. ARF displacements during HIFU therapy were shown to initially depend on attenuation before ablation, but once the temperature exceeded the ablation threshold, viscoelastic properties dominated. A second barrier for HIFU is the need to reliably deliver acoustic energy to the target. Nonlinear acoustic simulations were conducted using patient derived computed tomography (CT) data to evaluate the HIFU therapy in the kidney. The simulations revealed that attenuation and refraction contributed roughly equally to intensity loss. Focal splitting associated with refraction was found to be the main effect reducing the heating efficacy, and a method to employ phase correction at the source was proposed to mitigate the effect.

The Electromagnetic Acoustic (EMA) technique is a novel multi-modal technique for medical imaging. It is sensitive, in principle, to contrast in mechanical properties and electrical properties and has potential in a number of applications such as breast tumour detection where there will be contrast between diseased and healthy tissue and high intensity focused ultrasound monitoring, where there will be contrast when tissue is ablated. A complete computational model for the EMA imaging is developed. The model considers the linear or nonlinear propagation of ultrasound in soft tissue, the dynamic response of the viscoelastic soft tissue to acoustic radiation force (ARF) stimulation and scattering of electromagnetic waves with and without the Doppler effect. The suitability of the EMA imaging for breast tumour detection is evaluated, modelling a tumour as a spherical inclusion in an infinite homogeneous background tissue with clinically relevant material properties. The results show that variations of the mechanical properties of underlying healthy breast tissue and tumour tissue in clinically feasible range should result in a change in the amplitude of the first Doppler component (FDC) of up to 50%, and varying the electrical contrast leads to a change in the ratio of the FDC and unshifted component (UC) of less than 1 dB. The relative difference between the first Doppler component and the unshifted component is greater than 68 dB and therefore the frequency demodulation may pose a significant challenge if EMA imaging is used for breast tumour detection. The feasibility of using the EMA imaging for real-time monitoring of High Intensity Focused Ultrasound (HIFU) therapy is also investigated. Simulations conducted with realistic liver tissue properties show that the induced Doppler effect in the scattered EM wave is not well correlated with the growth of thermal lesion, therefore it is unlikely to be a good indicator of the lesion size. EMA imaging may not be appropriate for monitoring HIFU therapy.

High intensity focused ultrasound (HIFU) provides a wide range of noninvasive therapies ranging from drug delivery to the destruction of kidney stones. In particular, thermal ablation by HIFU presents an effective noninvasive method for the treatment of deep seated solid tumours. HIFU’s further uptake is limited by a need for improved treatment planning and monitoring. Two nonlinear acoustic phenomena that play key roles in HIFU treatment: finite amplitude effects that lead to the generation of harmonics and steepening of wavefronts, and acoustic cavitation. The former must be taken into careful consideration for treatment planning purposes, while the latter has the potential to provide fast, real-time, cost effective treatment monitoring. The first half of this thesis provides new measurements for the nonlinear acoustic properties of tissue, assesses the validity of two common modelling techniques for simulating HIFU fields. The second half develops a new method for combining passive acoustic mapping- an ultrasound monitoring technique- with MR thermometry, to assess estimates of cavitation enhanced heating derived from passive acoustic maps. In the first results chapter B/A was measured in ex-vivo bovine liver, over a heating/ cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique (FAIS), which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane-wave through liver and B/A was chosen so that numerical simulations matched measured waveforms. Results showed that attenuation initially decreased with heating then increased after denaturation, sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. These data disagree with other reports that show a significant change and suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity. In the second results chapter two common methods of modelling HIFU fields were compared with hydrophone measurements of nonlinear HIFU fields at a range of frequencies and pressures. The two methods usedwere the KZK equation and the commercial package PZFlex. The KZK equation has become the standard method for modelling focused fields, while the validity of PZFlex for modelling these types of transducers is unclear. The results show that the KZK equation is able to match hydrophone measurements, but that PZFlex underestimates the magnitude of the harmonics. Higher order harmonics in PZFlex are not the correct shape, and do not peak around the focus. PZFlex performs worse at higher pressures and frequencies, and should be used with caution. In the final two chapters a system for estimating cavitation-enhanced heating from acoustic maps is developed and benchmarked against magnetic resonance thermometry methods. The first chapter shows that the ultrasound and MR monitoring systems are compatible, and registers the two imaging systems. The HIFUfocus is clearly visible in passive maps acquired in the absence of cavitation and these coincide with the centre of heating in MR temperature images. When cavitation occurs, it coincides spatially and temporally with the appearance of a clear spike in temperature, especially when the passive maps are processed using the Robust Capon Beamformer algorithm. The final chapter shows how passive maps can be converted into thermal heating inputs, and used to estimate cavitation-enhanced temperature increases. These estimates have the potential to closely match maximum temperature rise, and estimated thermal dose after the estimated temperature rise is spatially averaged. However, themethod is not always successful. This is partly due to uncertainties in MR thermometry estimates, partly due to uncertainties in the acoustic properties of tissue.