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Background PanDox is a Phase-1 trial of chemotherapeutic drug delivery to pancreatic tumors using ultrasound-mediated hyperthermia to release doxorubicin from thermally sensitive liposomes. This report describes trial-related hyperthermia simulations featuring: (i) new ultrasonic properties of human pancreatic tissues, (ii) abdomen deflections imposed by a water balloon, and (iii) respiration-driven organ motion. MethodsPancreas heating simulations were carried out using three patient body models. Pancreas acoustic properties were varied between values found in the literature and those determined from our human tissue study. Acoustic beam distortion was assessed with and without balloon-induced abdomen deformation. Target heating was assessed for static, normal respiratory, and jet-ventilation-controlled pancreas motion. Results Human pancreatic tumor attenuation is 63% of the literature values, so that pancreas treatments require commensurately higher input intensity to achieve adequate hyperthermia. Abdominal wall deformation decreased the peak field pressure by as much as 3.5 dB and refracted the focal spot by as much as 4.5 mm. These effects were thermally counteracted by sidelobe power deposition, so the net impact on achieving mild hyperthermia was small. Respiratory motion during moving beam hyperthermia produced localized regions overheated by more than 8.0 °C above the 4.0 °C volumetric goal. The use of jet ventilation reduced this excess to 0.7 °C and yielded temperature field uniformity that was nearly identical to having no respiratory motion. Conclusion Realistic modeling of the ultrasonic propagation environment is critical to achieving adequate mild hyperthermia without the use of real time thermometry for targeted drug delivery in pancreatic cancer patients.

Introduction Many small molecules and biologic therapeutics have been developed for solid tumor therapy. However, the unique physiology of tumors makes the actual delivery of these drugs into the tumor mass inefficient. Such delivery requires transport from blood vessels, across the vasculature and into and through interstitial space within a tumor. This transportation is dependent on the physiochemical properties of the therapeutic agent and the biological properties of the tumor. It was hoped the application of nanoscale drug carrier systems would solve this problem. However, issues with poor tumor accumulation and limited drug release have impeded clinical impact. In response, these carrier systems have been redesigned to be paired with targetable external mechanical stimuli which can trigger much enhanced drug release and deposition. Areas covered The pre-clinical and clinical progress of thermolabile drug carrier systems and the modalities used to trigger the release of their cargo are assessed. Expert opinion Combined application of mild hyperthermia and heat-responsive liposomal drug carriers has great potential utility. Clinical trials continue to progress this approach and serve to refine the technologies, dosing regimens and exposure parameters that will provide optimal patient benefit.

The localization of microbubbles to a treatment site has been shown to be essential to their effectiveness in therapeutic applications such as targeted drug delivery and gene therapy. A variety of different strategies for achieving localization has been investigated, including biochemical targeting, acoustic radiation force, and the incorporation of superparamagnetic nanoparticles into microbubbles to enable their manipulation using an externally applied magnetic field. The third of these strategies has the advantage of concentrating microbubbles in a target region without exposing them to ultrasound, and can be used in conjunction with biochemical targeting to achieve greater specificity. Magnetic microbubbles have been shown to be effective for therapeutic delivery in vitro and in vivo. Whether this technique can be successfully applied in humans however remains an open question. The aim of this study was to determine the range of flow conditions under which targeting could be achieved. In vitro results indicate that magnetic microbubbles can be retained using clinically acceptable magnetic fields, for both the high shear rates (approx. 10(4) s(-1)) found in human arterioles and capillaries, and the high flow rates (approx. 3.5 ml s(-1)) of human arteries. The potential for human in vivo microbubble retention was further demonstrated using a perfused porcine liver model.

Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.

A theoretical model is sought to explain recent experimental results which have shown the sudden onset of inertial cavitation after a significant period of ultrasound insonation. It is thought that there initially exists a population of bubbles with sub-optimal radii for inertial cavitation, which grow during the insonation period to become nuclei within the optimal size range to cavitate inertially. Rectified diffusion during bubble oscillation, and the changes in the bubble nucleation environment and their subsequent dynamics caused by ultrasonic heating, are amongst the mechanisms that could be responsible for nuclei growth. In this work, the effects of elevating the temperature of the medium surrounding a bubble, resulting in higher vapour pressures and decreased solubility of gas in the bulk medium, is investigated. A single bubble model is formulated by coupling a numerical solution of the mass diffusion PDE and the Rayleigh-Plesset Equation, taking into account temperature dependence of surface tension, vapour pressure, Henry’s constant and diffusion coefficient. It is shown that an increase in the temperature at HIFU-relevant heating rates is on its own not sufficient to increase the radii of sub-optimal bubbles into the optimal range for inertial cavitation to take place.

Microbubble-enhanced sonothrombolysis is a promising approach to increasing the tolerability and efficacy of current pharmacological treatments for ischemic stroke. Maintaining therapeutic concentrations of microbubbles and drugs at the clot site, however, poses a challenge. The objective of this study was to investigate the effect of magnetic microbubble targeting upon clot lysis rates in vitro. Retracted whole porcine blood clots were placed in a flow phantom of a partially occluded middle cerebral artery. The clots were treated with a combination of tissue plasminogen activator (0.75 µg/mL), magnetic microbubbles (∼107 microbubbles/mL) and ultrasound (0.5 MHz, 630-kPa peak rarefactional pressure, 0.2-Hz pulse repetition frequency, 2% duty cycle). Magnetic targeting was achieved using a single permanent magnet (0.08–0.38 T and 12-140 T/m in the region of the clot). The change in clot diameter was measured optically over the course of the experiment. Magnetic targeting produced a threefold average increase in lysis rates, and linear correlation was observed between lysis rate and total energy of acoustic emissions.

The study of the effects of ultrasound-induced acoustic cavitation on biological structures is an active field in biomedical research. Of particular interest for therapeutic applications is the ability of oscillating microbubbles to promote both cellular and tissue membrane permeabilisation and to improve the distribution of therapeutic agents in tissue through extravasation and convective transport. The mechanisms that underpin the interaction between cavitating agents and tissues are, however, still poorly understood. One challenge is the practical difficulty involved in performing optical microscopy and acoustic emissions monitoring simultaneously in a biologically compatible environment. Here we present and characterise a microfluidic layered acoustic resonator (µLAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring and microscopy of biological samples. The µLAR facilitates in vitro ultrasound experiments in which measurements of microbubble dynamics, microstreaming velocity fields, acoustic emissions and cell-microbubble interactions can be performed simultaneously. The device and analyses presented provide a means of performing mechanistic in vitro studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

Ultrasound and microbubbles have been shown to accelerate the breakdown of blood clots both in vitro and in vivo. Clinical translation of this technology is still limited, however, in part by inefficient microbubble delivery to the thrombus. This study examines the obstacles to delivery posed by fluid dynamic conditions in occluded vasculature and investigates whether magnetic targeting can improve microbubble delivery. A two-dimensional computational fluid dynamic (CFD) model of a fully occluded Y-shaped microarterial bifurcation was developed to determine: (i) the fluid dynamic field in the vessel with inlet velocities from 1-100 mm/s (corresponding to Reynolds numbers 0.25-25); (ii) the transport dynamics of fibrinolytic drugs; and (iii) the flow behavior of microbubbles with diameters in the clinically-relevant range (0.6-5µm). In vitro experiments were carried out in a custom built microfluidic device. The flow field was characterized using tracer particles, and fibrinolytic drug transport was assessed using fluorescence microscopy. Lipid-shelled magnetic microbubbles were fluorescently labelled to determine their spatial distribution within the microvascular model. In both the simulations and experiments, the formation of laminar vortices and an abrupt reduction of fluid velocity were observed in the occluded branch of the bifurcation, severely limiting drug transport towards the occlusion. In the absence of a magnetic field, no microbubbles reached the occlusion, remaining trapped in the first vortex, within 350µm from the bifurcation center. The number of microbubbles trapped within the vortex decreased as the inlet velocity increased, but was independent of microbubble size. Application of a magnetic field (magnetic flux density of 76 mT, magnetic flux density gradient of 10.90T/m at the centre of the bifurcation) enabled delivery of microbubbles to the occlusion and the number of microbubbles delivered increased with bubble size and with decreasing inlet velocity.

A significant barrier to successful drug delivery is the limited penetration of nanoscale therapeutics beyond the vasculature. Building on recent in vivo findings in the context of cancer drug delivery, the current study investigates whether modification of nanoparticle drug-carriers to increase their density can be used to enhance their penetration into viscoelastic materials under ultrasound exposure. A computational model is first presented to predict the transport of identically sized nanoparticles of different densities in an ultrasonic field in the presence of an oscillating microbubble, by a combination of primary and secondary acoustic radiation forces, acoustic streaming, and microstreaming. Experiments are then described in which near monodisperse (polydispersity index < 0.2) nanoparticles of approximate mean diameter 200 nm and densities ranging from 1.01 g/cm3 to 5.58 g/cm3 were fabricated and delivered to a tissue-mimicking material in the presence or absence of a microbubble ultrasound contrast agent, at ultrasound frequencies of 0.5 MHz and 1.6 MHz and a peak negative pressure of 1 MPa. Both the theoretical and experimental results confirm that denser particles exhibit significantly greater ultrasoundmediated transport than their lower density counterparts, indicating that density is a key consideration in the design of nanoscale therapeutics.

A new formulation of volatile nanodroplets stabilized by a protein and polymer coating and loaded with magnetic nanoparticles is developed. The droplets show enhanced stability and phase conversion efficiency upon ultrasound exposure compared with existing formulations. Magnetic targeting, encapsulation, and release of an anticancer drug are demonstrated in vitro with a 40% improvement in cytotoxicity compared with free drug.

Less than 10% of drug candidates successfully pass through clinical trials and gain regulatory approval. One of the causes of translational failure is poor drug pharmacokinetics which are inadequately predicted by preclinical studies. Despite improvements in pharmacokinetic prediction in recent years, the circulating kinetics of many novel biologic agents, nanomedicines and drug delivery systems which exhibit transporter-mediated clearance, and size-dependent uptake by the reticuloendothelial system (RES), do not scale between species in a predictable manner and suffer with inadequate delivery to the therapeutic target. This hinders clinical translation, thus highlighting the need for more accurate preclinical testing to predict human drug disposition and facilitate improved drug delivery. Normothermic machine perfusion (NMP) is a method of organ preservation whereby oxygenation and nutrient provision to an organ is enabled via circulation of a cellular or acellular perfusate at body temperature. This maintains organs in a quasi-physiological state which we hypothesise will preserve the pharmacokinetic processes of drug distribution, metabolism and excretion, reflecting drug disposition in a close-to-man environment. The primary aim of this thesis is to determine whether human and porcine NMP clearance organs (kidney, spleen and liver) serve as a tool for predicting human drug clearance and for assessing drug delivery. Perfusion protocols were developed to support prolonged perfusion of i) human NMP liver and kidney, ii) laboratory-procured, porcine NMP liver and kidney and iii) abattoir-derived, porcine NMP liver and spleen. Systematic comparison of the different approaches demonstrated a robust physiological platform for profiling therapeutic pharmacokinetics and delivery. A small molecule therapeutic drug was delivered to the human and laboratory-procured, porcine liver and kidney models with assessment of pharmacokinetics. Both human and porcine organs were able to successfully support distribution, metabolism, hepatobiliary and renal excretion of the drug. Organ specific and total body clearance were calculated using physiological and pharmacokinetic data from the NMP organs and compared to human clinical data. Human NMP organs, which are discarded for transplantation with varying degrees of injury, did not accurately predict human clearance. However, pristine porcine NMP liver and kidney kidneys enabled accurate prediction of human intrinsic organ and total body clearance; the most important measure used to establish the safe dosing regimen when translating a novel drug into patients. This represents the first successful validation of a large mammalian normothermically preserved organ model against clinical data for an approved drug. Finally, the NMP spleen, liver and kidney were used to investigate the kinetics and delivery of a model polymeric nanoparticle and a viral vector; two classes of agent commonly captured by the RES and poorly translated from bench to bedside. PEGylated and non-PEGylated nanoparticle kinetics measured in NMP organs correspond to existing small animal data and provide evidence for nanoparticle capture in large animal organs for which data is limited. The NMP porcine liver was also able to support the circulating kinetics and integration of a viral vector into host hepatocyte DNA; a first for drug delivery studies in the ex vivo NMP liver and an exciting prospect for the delivery of hepatocyte-targeted gene therapies. In conclusion, despite limitations such as the effect of ischaemia reperfusion injury on organ physiology and the low throughput nature of the technology, this work demonstrates the utility of NMP clearance organs as a tool for predicting human pharmacokinetics and assessing drug delivery. The richness of data generated from the models has the potential to serve as an adjunct to existing preclinical technologies, for accelerating the development of novel therapeutics for patient benefit.

Liver transplantation is the only definitive treatment for end-stage liver disease. However, a shortage of suitable donor organs means that currently up to 30% of patients become too ill or die before ever receiving a transplant. Normothermic machine perfusion (NMP) offers a potential solution by reducing preservation injury which might enable the safe transplantation of higher-risk livers, thereby expanding the donor pool and reducing waiting list mortality. To achieve the full potential of machine perfusion in transplantation, a set of sensitive and specific biomarkers are needed to enable accurate prediction of a donated livers ability to survive the transplantation process and function in the recipient. Unfortunately, such a set of biomarkers is not yet established. This thesis explores the potential of magnetic resonance (MR) imaging and spectroscopy to provide novel biomarkers for the prediction of transplantability in ex vivo livers. Deceased donor transplantation necessarily subjects livers to a metabolic and physiological insult during death of the donor. I quantified this using magnetic resonance in porcine models. Then I assessed the ability of temperature-corrected MR techniques to assess organ viability in ex vivo human liver during the traditional preservation of static cold storage. I then demonstrated non-invasive assessment of porcine livers during prolonged perfusion on a novel MR-compatible normothermic machine perfusion (NMP) system. Finally, I showed the proof-of-principle for performing MR assessment of ex vivo human livers during NMP. Transplantation is currently undergoing a dramatic transformation with the incorporation of machine perfusion technologies and this thesis suggests that MR techniques hold immense potential for providing non-invasive assessment of transplantability in ex vivo livers.

Tumour and tumour microenvironment exploitation of immunologic checkpoints like PD-L1 can enable immune evasion. Recent advances in oncology, have brought forward antibody drugs, such as anti-PD-L1, capable of blocking and/or mediating the eradication of cells that express PD-L1. Despite promise across a wide range of indications, clinically such agents meaningfully extend survival only in a fraction of patients treated. Immature, heterogenous blood vessels, elevated interstitial pressures, and stromal barriers severely limit the accumulation and extravasation of drugs within the tumour microenvironment. We hypothesize that the physical size of antibody therapeutics, such as anti-PD-L1, compounded by delivery barriers could be a factor limiting their efficacy. Recently, submicron cavitation nucleation agents, sonosensitive particles (SSPs), activated by focused ultrasound have been shown to improve the delivery of large, anti-cancer therapeutics via microstreaming-mediated transport. Our aim was to pre-clinically investigate whether systemically-dosed anti-PD-L1 followed by SSPs and the application of focused ultrasound could improve the intratumoural delivery and therapeutic efficacy of anti-PD-L1. Efficacy studies revealed that pairing anti-PD-L1 with ultrasound-mediated cavitation can significantly extend median survival time. Cavitation was also found to enhance the intratumoural accumulation of anti-PD-L1 and three other antibodies. Further, the binding intensity and distribution of anti-PD-L1 improved in cavitation-treated tumours. Immunological analysis suggests that anti-PD-L1 paired with cavitation enables the innate and adaptive immune system to leverage the modest cavitation-induced inflammation and improved anti-PD-L1 delivery to facilitate tumour attack. Taken in a broader context, this pre-clinical work suggests that ultrasound-mediated cavitation warrants further investigation as viable tool to improve drug delivery clinically.

Injections are a commonly used and widely accepted method of drug administration to prevent and treat diseases. Although using a needle and syringe provides a rapid, lowcost, and direct way of administering almost any kind of therapeutic agent into the body, it is a painful and invasive method leading to issues of patient compliance and needle–phobia. The delivery of drugs through the skin (transdermal delivery) is an attractive alternative approach because it is non-invasive and has low associated risks. However, the penetration of molecules into/through the skin is severely restricted by the outermost layer of the skin (stratum corneum). Initial studies of ultrasound-mediated, cavitation-enhanced transdermal drug delivery have shown great promise by utilising a gel formulation containing polymeric cavitation nuclei to help deliver a model immunogen (Ovalbumin) into the skin. However, enhanced adoption of this technology to permit delivery of several classes of therapeutic agents requires further validation and optimisation and is currently stymied by the lack of real-time monitoring when used across a broad range of therapeutic classes and environmental conditions. Such monitoring is especially important in situations where the dose of the delivered therapeutic agent needs to be precisely defined. Therefore, the focus of this thesis is to achieve transdermal delivery using cavitation-enhanced transport and develop methodologies to monitor or at least confirm successful delivery in real-time. [abstract continued in thesis]

The thesis describes the development of a transportable normothermic kidney preservation device which is able to perfuse discarded human and porcine kidneys for up to 24 hours for the very first time. Recirculation of the urine to facilitate maintenance of perfusate volume and homeostasis is a novel approach. Utilisation of this perfusion device could enable viability assessment, decrease organ discard rate and improve transplantation logistics. Thirty-six clinically declined human kidneys were perfused, 19 from donors after brain stem death and 17 from donors after circulatory death: n=4 kidneys were perfused using Ringer’s lactate to replace excreted urine volume, and n=32 using urine recirculation. In all cases, normothermic perfusion either maintained or slightly improved the histopathologically assessed tubular condition, achieved physiological arterial flow levels and effective urine production. Biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and liver fatty acid-binding protein (L-FABP) were detected and quantified in the perfusates with and without urine recirculation. NGAL could be promising for clinical use, as it was inversely correlated with median arterial flow. Only kidneys with urine recirculation were readily perfused up to 24 hours and exhibited physiological perfusate sodium levels, whilst kidneys without urine recirculation achieved reduced normothermic perfusion time and significantly higher perfusate sodium. Metabolomics analyses of perfusate samples detected urea and two sugars as the main difference between kidneys with and without urine recirculation. Proteomics analyses not only revealed that human kidneys have become metabolically active during normothermic perfusion, but they also detected that damage-associated molecular patterns known to contribute to ischaemia-reperfusion-injury, were significantly downregulated in biopsies from kidneys with urine recirculation compared to kidneys without. As a final part, porcine perfusion studies were performed with the aim to revisit the findings obtained during the human experiments and to render a valid comparison between urine recirculation and urine replacement in undamaged kidneys.

Cancer imposes a significant disease burden and constitutes a major societal challenge. Despite being widely used, chemotherapy suffers numerous disadvantages, including off- target drug toxicity, poor tumour penetration, and drug resistance. The use of ultrasound in combination with contrast agents has shown promise in enhancing outcomes in the treatment of cancerous and non-cancerous diseases. However, the underlying biophysical processes that underpin their interactions with tissues remain poorly understood. The aim of the research presented in this thesis is to develop methods to elucidate these processes. Development is hindered by the difficulties involved in isolating cellular parameters, noninvasively quantifying biological features at the molecular scale, and recreating a predictable and repeatable ultrasound field. To overcome these challenges, giant unilamellar vesicles made with 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol were used initially as cell models and exposed to therapeutically relevant conditions in a purpose-built acoustofluidic device. The resulting effects on the hydration and permeability of the vesicle membranes and the dynamics of their intravesicle milieu were characterised in situ using quantitative microscopy. Results show that flow, ultrasound, and microbubbles led to an increase in vesicle membrane hydration, while dehydration was seen only in the presence of microbubbles. An increase in permeability was observed for all exposure conditions, and was accentuated when microbubble shell fragments were incorporated in the vesicle bilayer. These findings thus indicate that ultrasound-mediated delivery is governed by a combination of physical and chemical mechanisms that influence the permeation of molecules through lipid membranes. Further, it was shown that exposure to the same conditions led to the onset of streaming flows within the vesicles’ lumen. An experimental proof-of-concept for extending the study in vitro was also presented. Actuating the motion of cytoplasmic constituents may potentially induce a broad range of underexplored and therapeutically relevant bioeffects.

Cancer therapeutics are limited in efficacy by poor penetration into tumour tissue. This doctoral thesis aims to explore how changing the density of a therapeutic could influence its ultrasound-mediated delivery and penetration depth, thereby testing the hypothesis that increasing particle density increases penetration depth of nanoparticles under ultrasound exposure. Density-enhanced transport in the presence of ultrasound-induced cavitation is studied utilising a combination of numerical simulations, in vitro and in vivo experimental models. The penetration depth of nanoparticles of different densities was first predicted using a computational model to assess the magnitude of acoustic and fluid dynamic forces on a spherical nanoparticle in the presence or absence of a cavitating microbubble. This simulation showed that a denser particle will be transported further when exposed to ultrasound. Furthermore, cavitation was identified as fundamental for enhanced transport, with forcing due to microstreaming dominating the transport behaviour. These predictions were validated and supported experimentally using an in vitro model, where a fivefold increase in particle density resulted in a 43 % increase in peak particle penetration depth. To form a closer analogue to the tumour environment, a three-dimensional flow-vessel phantom was then implemented, where the penetration depths of three density-contrasting nanoparticles with unique fluorescent tags – co-delivered with either a micro- or a nano-scale cavitation agent – were studied. A trend between nanoparticle density and extravasation depth was again discernible, with the densest particles penetrating the furthest. However, the choice of cavitation agent often had a greater influence on particle transport. Finally, experiments were performed in a tumour-bearing mouse model, where the bio-distribution of fluorescent nanoparticles was assessed after ultrasound exposure. A bistatic HIFU setup was used to activate cavitation agents, and passive acoustic mapping was implemented to detect cavitation activity within the tumour site. Density-contrasting fluorescent nanoparticles were used as mock therapeutics, and were successfully detected in ex vivo tumours through IVIS imaging. However, due to experimental limitations, the influence of nanoparticle density on ultrasound-mediated drug delivery in vivo could not be determined, and so recommendations are made for how the hypothesis can be further validated.

High intensity focused ultrasound (HIFU) is an emerging non-invasive thermal ablative modality that can be utilised for the treatment of solid organ tumours, including liver cancer. Acoustic cavitation is a phenomenon that can occur during HIFU and its presence can enhance heating rates. One major limitation of thermal ablative techniques in general, such as radiofrequency and microwave ablation, is the heat sink effect imparted by large vasculature. Thermal advection from blood flow in vessels ≥ 3 – 4 mm in diameter has been shown to significantly reduce heating rates and peak temperatures in the target tissue, potentially leading to treatment failure. With regards to HIFU therapy, a clearer understanding is required of the effects of blood flow on heating, cavitation and thermal tissue necrosis, which is the treatment endpoint in clinical thermal ablation. Therefore, the overall aim of this thesis project was to elucidate the effects of blood flow on HIFU-induced heating, cavitation and histological assessment of thermal ablation. A unique isolated, perfused porcine liver model was used in order to provide a relevant test bed, with physiological and anatomical characteristics similar to the in vivo human liver. The normothermic liver perfusion device used in all studies presented in this work can keep an organ alive in a functional state ex vivo for in excess of 72 hours. A further advantage of the liver perfusion device was that it allowed blood flow to be stopped completely and resumed rapidly, allowing studies to be conducted under zero flow conditions. A therapeutic HIFU system was used in order to deliver HIFU therapy to regions of hepatic parenchyma adjacent (≤ 3 mm) to large (≥ 5 mm) blood vessels or away from vasculature (≥ 1 cm) at either 1.06 MHz or at 3.18 MHz. Cavitation events during HIFU therapy were spatio-temporally monitored using a previously developed passive acoustic mapping (PAM) technique. The cavitation threshold at each frequency was determined through assessment of acoustic emissions acquired through PAM during HIFU exposure at a range of acoustic pressures. Real time thermal data during HIFU therapy were obtained using an implantable 400 μm thermocouple, aligned with the HIFU focus, in order to assess the effect of large vessel blood flow on peak tissue temperatures. Thermal data were obtained at 1.06 MHz, in the presence of acoustic cavitation and at 3.18 MHz, in the absence of cavitation, both in the presence and complete absence of blood flow. Finally, histological assessment of cell viability and cell death was performed in order to determine whether any heat sink effect could be overcome, with the achievement of complete tissue necrosis in treatment regions directly adjacent to large vasculature. This work demonstrated for the first time that in perfused, functional liver tissue, the presence of large vasculature and physiological blood flow does not significantly affect ablative HIFU therapy, both in terms of peak focal tissue temperatures attained and histological evidence of complete tissue necrosis. Therefore, HIFU may be superior to other ablative modalities in treating tumours in tissue regions adjacent to major vascular structures, but further work needs to be performed to correlate the experimental findings with clinical outcomes.

Despite therapeutic advances, ischaemic stroke remains one of the leading causes of mortality and disability worldwide. Microbubble-enhanced sonothrombolysis is an advantageous method to accelerate the reopening of occluded arteries, but further improvements in safety and efficacy are required to attain widespread clinical implementation. This thesis investigates the potential of magnetically targeted microbubbles to enhance sonothrombolysis. First, a computational and experimental model of a thrombosed vascular network was developed. It showed that magnetic targeting considerably increases the number of microbubbles delivered to the thrombus, a key element for effective sonothrombolysis. The effect of these magnetically targeted microbubbles on rates of thrombus breakdown was then investigated in vitro, first with ultrasound and microbubbles alone (mechanical sonothrombolysis), then with the addition of fibrinolytic drugs (enzymatic sonothrombolysis). Magnetic targeting was not found to affect mechanical treatment, and mechanical lysis rates were highly variable. On the other hand, magnetic targeting significantly accelerated enzymatic treatment, even with low concentrations of fibrinolytic drugs. The mechanisms underlying both treatments were discussed. In addition, treatment safety was examined. Specifically, the risk of secondary embolism and the feasibility of real-time treatment monitoring were assessed. Neither treatment increased the risk of downstream vascular embolism with large clot fragments. Acoustic monitoring of enzymatic sonothrombolysis was feasible, as a strong correlation was found between lysis rates and the energy of acoustic emissions near the clot surface. There was no strong correlation in mechanical sonothrombolysis. Magnetic targeting may therefore be regarded as a promising adjuvant to enzymatic sonothrombolysis, improving microbubble delivery to the clot, accelerating its breakdown, and reducing injected drug doses. It is hoped that targeted treatment and real-time monitoring will enhance safety and efficacy in the management of acute and chronic thrombosis, and ultimately improve patient outcomes.

High Intensity Focused Ultrasound (HIFU) is rapidly emerging as a viable alternative to more invasive methods for routine treatment of malignant tissue. Key to the success of HIFU therapy is the development of standardised Quality Assessment (QA) procedures, designed to regularly assess the safety and efficacy of clinical HIFU transducers. Current HIFU QA procedures offer either accurate spatial calibration at the expense of impractically long acquisition times, or more rapid characterisation that provides only a spatially averaged measure of performance. Exploitation of acoustically induced inertial cavitation, however, offers the potential for rapid, pre-treatment spatial calibration without the need for time consuming scanning routines. In this thesis, the concept of cavitation-based HIFU QA is validated for the first time, with various techniques developed to enable its implementation. To begin with, two-dimensional mapping using an array of needle hydrophones establishes that a growing cavitation region may be reconstructed at increasing insonation amplitudes. Extending the study to include a diagnostic linear array provides improved spatial resolution, enabling accurate, two-dimensional field characterisation within a novel, microparticle-seeded test medium that is shown to maximise the magnitude and spatial uniformity of cavitation occurrences. Performing this procedure in multiple focal planes provides a practical means of characterising a clinical HIFU transducer using readily available hardware. An alternative array geometry is proposed for rapid, three-dimensional field characterisation. To allow accurate placement of a large number of elements within such an array, a novel, multiple-layer Printed Circuit Board (PCB) construction method is adopted. A model is developed to predict the effect of the multiple acoustic layers of the array on its receive sensitivity spectrum and the results contrasted with both a finite element solution and experimental data to inform the eventual array design. An ideal passive array is thence specified through a combined experimental and computational approach, taking care to optimise the element size, number and distribution. This array configuration has the potential to enable reconstruction of a wide array of sources. The second generation prototype fulfils most of the requirements but has highlighted issues with crosstalk. Nevertheless, the results presented thus far provide a strong foundation for future work into cavitation-based HIFU QA.

Passive acoustic mapping (PAM) is a novel method for monitoring ultrasound therapies by mapping sources of acoustic activity, and in most cases cavitation activity, using an array of detectors. Although the range of its applications is indicative of its great potential, clinical adoption is currently hindered by its limited spatial resolution and the inherent difficulty of distinguishing, at depth, between nonlinear signals arising from nonlinear propagation and those arising from processes such as cavitation. The objective of this thesis is to address this limitation, by improving both the detection of the signal-of-interest and the source localisation. An optimum data-adaptive array beamforming algorithm is proposed, Robust Beamforming by Linear Programming (RLPB), which exploits the higher-orderstatistics of the recorded signals, aiming at improving PAM source localisation. Both simulations and in vitro experimentation demonstrated improvement in PAM spatial resolution compared to a previously introduced algorithm, Robust Capon Beamformer. More specifically, under the in vitro conditions examined here, a 22% and 14% increase in the axial and transverse PAM resolution is respectively achieved. In terms of reliable signal-of-interest detection amongst interfering signals, a time-domain data-adaptive parametric model, Sum-of-Harmonics (SOH) model, is developed. This model enables accurate estimation of time-varying-amplitude narrowband components in the presence of broadband signals. Respectively, it can recover a weak broadband signal in the presence of a dominant narrowband component. Compared to conventional comb filtering, SOH model enables PAM of cavitation sources that better reflect their physical location and extent. PAM performance enhancement achieved by combining the proposed beamforming and filtering approaches is assessed in a context where spatial resolution really matters, namely for distinguishing between cavitation activity occurring inside a channel and perivascularly following cavitation-mediated extravasation. Adoption of the proposed method results in more accurate isolation of the broadband emissions from inertially cavitating sources, and more reliable localisation of these sources despite the long source-to-array distance has been observed. Such an improvement to the spatial accuracy of PAM paves the way towards its clinical translation, and in vivo experimentation is the next step for further validation of PAM in conjunction with the proposed methods under clinically relevant conditions.

The TARDOX study is a Phase I first-in-man proof-of-concept study which aims to demonstrate the safety and feasibility of targeted delivery of a thermo-sensitive liposome in combination with mild hyperthermia induced using an extracorporeal Focused Ultrasound (FUS) device. ThermoDox® is a specially formulated long-circulating and thermosensitive liposome, activated by conditions of mild hyperthermia, to release its cytotoxic doxorubicin content. FUS offers non-invasive and non-ionising approach to tumour therapy, classically by the mechanism of thermal ablation (High Intensity Focused Ultrasound, HIFU) rather than hyperthermia. Nine patients have received intervention; a single treatment cycle of ThermoDox® combined with FUS to a single target primary or secondary liver tumour using an ultrasound-guided extracorporeal FUS device (Model JC200 Focused Ultrasound Tumor Therapeutic System, Haifu Medical). Five cases proceeded with real-time thermometry and four without. In those with thermometry, optimal hyperthermia (T>39.5°C for over 300 cumulative and 60 continuous seconds, and CEM43 < 60 minutes) was achieved in 4/5. Core tumour biopsies were used for drug quantification studies using high performance liquid chromatography. In an interim analysis, of the evaluable tissue samples analysed to date, an average of 8.41 μg/g ± 4.37 SD of intratumoral doxorubicin was demonstrated in those receiving optimal hyperthermia under real-time thermometry (n=2) and 6.75 μg/g ± 0.13 SD in those whose intervention proceed without thermometry (n=2). Overall, an aver- age of 7.58 μg/g ± 2.70 SD of doxorubicin was seen in the included post-delivery samples analysed to date (n=4). This compares to an average of 2.48 μg/g ± 1.26 SD in evaluable samples taken immediately prior to FUS-mediated delivery (n=4). Microscopic analysis of post-delivery biopsies demonstrated intercalation of doxorubicin in the nuclei of tumour cells in 6/6 patients with adequate tissue samples, evidencing that release of bioavailable drug from the administered liposomes has been achieved. Radiological follow-up at two weeks with 18F-FDG PET-CT demonstrated unequivocal localised response in 3/4 evaluable patients receiving optimal hyperthermia and 2/4 without real-time thermometry (subjective response in 1 of the remaining 2), despite only receiving a single standard dose treatment cycle of doxorubicin. Serious adverse events were limited to transient neutropenia in 4/9 patients, in line with the adverse event profile for doxorubicin, and prolonged hospital stay due to confusion in 1/9. We have demonstrated that combined treatment with ThermoDox® and extracorporeal targeted FUS hyperthermia is feasible, safe and can enhance intratumoral drug delivery, providing localised response in human liver tumours refractory to standard chemotherapy. This study, which builds on decades of research efforts in targeted liposome delivery using FUS, is one of the earliest studies to translate ultrasound-mediated tumour targeting into the clinic. The use of chemotherapy in combination with FUS devices, as a non-invasive targeting method, has the potential to transform future clinical treatments for solid organ cancers.

One of the major limiting factors of cancer therapy is the inability to deliver a sufficient dose of drugs to a target tumour without incurring significant side effects. Recent advances in nano-sized liposome formulations have enabled encapsulation of anti-cancer agents that simultaneously reduce off-target toxicity and allow increased accumulation in tumours due to the enhanced permeability and retention effect. Yet, a method for reliably releasing the drug from encapsulation has yet to be found. In this work, low-temperature sensitive liposomes, which release an encapsulated anti-cancer agent (doxorubicin) in the temperature range of 39-45±C, are combined with focused ultrasound-induced mild hyperthermia to deliver a predictable, site-specific, and high dose of anti-cancer agents that has the potential of overcoming the current limitations of targeted drug delivery for cancer. To reach this aim, acoustic cavitation-enhanced heating was harnessed to produce rapid and efficient temperature rises that could be spatiotemporally monitored and eventually controlled. For this purpose, a reproducible, non-exothermic, cell-embedding tissue-mimicking model was first developed to have an attenuation coefficient, thermal response and cavitation threshold similar to liver. The phantom platform was then utilised to allow for high-throughput optimisation of ultrasound exposure parameters that efficiently and safely produced mild hyperthermia and drug release. Inertial cavitation-enhanced heating was successfully shown to enhance drug release from the liposomes and subsequent cancer cell death due to release of the encapsulated agents, using a 25% Duty Cycle, 1Hz PRF 6 s sonication sequence. The remotely detectable acoustic emissions associated with cavitation were then exploited to enable real-time monitoring and spatial mapping of drug release using a novel passive acoustic mapping method. Finally, these developments and techniques were combined to demonstrate the feasibility of cavitation enhanced drug release and delivery in a world-unique isolated normothermic liver perfusion model that provides blood-flow rates comparable to those encountered in humans.

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.

Cavitation is a phenomenon present during many ultrasound therapies, including the thermal ablation of malignant tissue using high intensity focused ultrasound (HIFU). Inertial cavitation, in particular, has been previously shown to result in increased heat deposition and to be associated with broadband noise emissions that can be readily monitored using a passive receiver without interference from the main ultrasound signal. The present work demonstrates how an array of passive receivers can be used to generate maps of cavitation distribution during HIFU exposure, uncovering a new potential method of monitoring HIFU treatment. Using a commercially available ultrasound system (z.one, Zonare, USA), pulse transmission can be switched off and data from 64 elements of an array can be simultaneously acquired to generate passive maps of acoustic source power. For the present work, a 38 mm aperture 5-10 MHz linear array was used, with the 64 elements chosen to span the entire aperture. Theory and simulations were used to show the spatial resolution of the system, the latter showing that the broadband nature of inertial cavitation makes passive maps robust to interference between cavitating bubbles. Passive source mapping was first applied to wire scatterers, demonstrating the ability of the system to resolve broadband sources. With the array transversely placed to the HIFU axis, high-resolution passive maps are generated, and emissions from several cavitating bubbles are resolved. The sensitivity of passive mapping during HIFU exposure is compared with that of an active cavitation detector following exposure. The array was then placed within a rectangular opening in the centre of the HIFU transducer, providing a geometric setup that could be used clinically to monitor HIFU treatment. Cavitation was instigated in continuous and disjoint regions in agar tissue mimicking gel, with the expected regions of cavitation validating the passive maps obtained. Finally, passive maps were generated for samples of ox liver exposed to HIFU. The onset of inertial cavitation as detected by the passive mapping approach was found to provide a much more robust indicator of lesioning than post-exposure B-mode hyperecho, which is in current clinical use. Passive maps based on the broadband component of the received signal were able to localize the lesions both transversely and axially, however cavitation is generally indicated 5 mm prefocal to the lesions. Further work is needed to establish the source of this discrepancy. It is believed that with use of an appropriately designed cavitation detection array, passive mapping will represent a major advance in ultrasound-guided HIFU therapy. Not only can it be utilized in real-time during HIFU exposure, without the need to turn the therapeutic ultrasound field off, but it has also been shown in the context of the present work to provide a strong indicator of successful lesioning and high signal-to-noise compared to conventional B-mode ultrasound techniques.

Side effects of current chemotherapeutics limit their use in cancer therapy. Although many current drugs are highly toxic and potent, the effects they have on non-cancerous tissue are unbearable for patients. Targeting these drugs may provide a means to restrict their toxic effects to only cancer tissue while leaving healthy tissue unaffected. This approach requires that the drug is only available in cancer tissue, which has been achieved here by encapsulating drugs into liposomal nano-capsules which are capable of passively accumulating in cancerous tissue via the enhanced permeability and retention effect (EPR). In addition to localisation, a threshold dose must be achieved to deliver the desired toxic effect to the target tumour tissue. Previous strategies have relied on passive 'leaching' of the drug from liposomes, however this 'leaching' does not necessarily achieve the threshold dose required. In the present work, a new generation of liposomes has been developed whereby release is solely achieved in the presence of ultrasound triggered cavitation. Instigation of such cavitation events would normally require the target tissue be exposed to high and possibly damaging ultrasound pressures. To remove the need for these high pressures, cavitation nuclei have been developed to lower the cavitation threshold of surrounding media. To allow for improved co-localisation and treatment deeper into cancer tissue, cavitation nuclei were developed to be in the nanoscale size range. Two types of novel cavitation nuclei were produced, a rough surfaced carbon nanoparticle (CNP, ~180 nm) and smooth shaped polymeric nano-cup particle (NC, ~150, 470, or 770 nm). Both types of particle are solid nanoparticles with gas entrapped on their surface which was capable of cavitating in response to ultrasound without greatly affecting the particle itself. These particles are classified as cavicatalytic nanoparticles due to their ability to reduce the cavitation threshold of their surrounding media without being destroyed themselves. Finally, an entirely nanoscale release system was developed and tested in vitro and in vivo. The drug carrier (the liposome) and effector agent (the cavicatalytic nanoparticle) were used to demonstrate ultrasound triggered drug release, specifically in response to the generation of cavitation events. These cavitation events could be non-invasively monitored and characterised, adding to the potential clinical utility of the technologies developed and described here.

Poor drug penetration through tumor tissue has emerged as a fundamental obstacle to cancer therapy. The solid tumor microenvironment presents several physiological abnormalities which reduce the uptake of intravenously administered therapeutics, including leaky, irregularly spaced blood vessels, and a pressure gradient which resists transport of therapeutics from the bloodstream into the tumor. Because of these factors, a systemically administered anti-cancer agent is unlikely to reach 100% of cancer cells at therapeutic dosages, which is the efficacy required for curative treatment. The goal of this project is to use high-intensity focused ultrasound (HIFU) to enhance drug delivery via phenomena associated with acoustic cavitation. ‘Cavitation’ is the formation, oscillation, and collapse of bubbles in a sound field, and can be broadly divided into two types: ‘inertial’ and ‘stable’. Inertial cavitation involves violent bubble collapse and is associated with phenomena such as heating, fluid jetting, and broadband noise emission. Stable cavitation occurs at lower pressure amplitudes, and can generate liquid microstreaming in the bubble vicinity. It is the combination of fluid jetting and microstreaming which it is attempted to explore, control, and apply to the drug delivery problem in solid tumors. First, the potential for cavitation to enhance the convective transport of a model therapeutic into obstructed vasculature in a cell-free in vitro tumor model is evaluated. Transport is quantified using post-treatment image analysis of the distribution of a dye-labeled macromolecule, while cavitation activity is quantified by analyzing passively recorded acoustic emissions. The introduction of exogenous cavitation nuclei into the acoustic field is found to dramatically enhance both cavitation activity and convective transport. The strong correlation between inertial cavitation activity and drug delivery in this study suggested both a mechanism of action and the clinical potential for non-invasive treatment monitoring. Next, a flexible and efficient method to simulate numerically the microstreaming fields instigated by cavitating microbubbles is developed. The technique is applied to the problem of quantifying convective transport of a scalar quantity in the vicinity of acoustically cavitating microbubbles of various initial radii subject to a range of sonication parameters, yielding insight regarding treatment parameter choice. Finally, in vitro and in vivo models are used to explore the effect of HIFU on delivery and expression of a biologically active adenovirus. The role of cavitation in improving the distribution of adenovirus in porous media is established, as well as the critical role of certain sonication parameters in sustaining cavitation activity in vivo..., ...it is shown that following intratumoral or intravenous co-injection of ultrasound contrast agents and adenovirus, both the distribution and expression of viral transgenes are enhanced in the presence of inertial cavitation. This ultrasound-based drug delivery system has the potential to be applied in conjunction with a broad range of macromolecular therapeutics to augment their bioavailability for cancer treatment. In order to reach this objective, further developmental work is recommended, directed towards improving therapeutic transducer design, using transducer arrays for treatment monitoring and mapping, and continuing the development of functionalized monodisperse cavitation nuclei.

High-intensity focused ultrasound (HIFU) is rapidly emerging as a viable alterna- tive to conventional therapies in the treatment of deep-seated, solid tumours. In contrast to surgical methods, extracorporeal HIFU transducers non-invasively tar- get pathogenic tissue deep beneath the skin, inducing thermal necrosis of a volume of tissue typically coincident with the ultrasound focus. More recently, cavitation activity has been observed to enhance focal heating, whilst providing a unique op- portunity for real-time treatment monitoring. Unfortunately, the stochastic nature of cavitation makes it difficult to initiate and sustain the level of cavitation activity required for enhanced heating, and to confine the spatial extent of cavitation to the focal volume. The overall aim of this thesis is to design and implement a real-time, closed- loop controller for sustaining thermally relevant cavitation within the HIFU focal region. This is intended to improve the speed and reproducibility of tissue ablation, whilst providing clinicians with real-time feedback as to the extent and location of the ablated region. A quantitative relationship between the level of cavitation activity and asso- ciated temperature rise is first sought experimentally, by investigating cavitation- enhanced heating in two different tissue-mimicking materials (TMM) that yield dif- ferent levels of cavitation for the same HIFU exposure conditions. It is found that a minimum level of inertial cavitation activity is required for cavitation-enhanced heating to dominate the heating process, which is achieved in the first material but not the second. However, the introduction of exogenous, artificial nuclei to the second material is seen to augment cavitation levels to the extent that cavitation- enhanced heating becomes dominant. Subsequently, HIFU experimentation is extended to non-perfused, ex vivo bovine liver, into which a variety of cavitation nuclei are introduced to augment cav- itation levels, and hence heating. Commercially available lipid-shelled microbub- bles are contrasted with custom-made sonosensitive nanoparticles for their ability to seed cavitation events, culminating in an empirical relationship between iner- tial cavitation and heating that is common to both types of exogenous nuclei, and which agrees with the in vitro results. Moreover, the abnormally large lesions pro- duced are found to correlate with a broad spatial distribution of inertial cavitation events, as seen on two-dimensional passive acoustic maps. Based on these encouraging results, a novel negative-feedback, real-time con- trol system is implemented to sustain inertial cavitation within the focal region for extended periods of time. The controller is designed to be both asymmetric and adaptive, deploying different feedback gains to adjust the peak rarefactional focal pressure (PRFP), depending on whether cavitation activity is above or below the level required for cavitation-enhanced heating. With active cavitation control in vitro, the associated focal temperature elevation is maintained at a cytotoxic level for 20 seconds using less than half the energy input required in the absence of cavi- tation control. In order to test the applicability of the novel controller to a near-physiological environment, HIFU exposures are eventually performed in a unique normothermic perfused liver model that accounts for both heat advection and nuclei replenish- ment. Following preliminary experimentation, the controller is modified to account for the inherent variability in the cavitation threshold of perfused tissue, whilst the cavitation demand is also increased to account for heat advection. Following these modifications, use of the controller is found to enable greatly improved re- producibility of HIFU-induced lesions compared to those achieved without cavita- tion control, with a lesion size that is directly related to the cavitation demand. A cost-effective method for enabling caviation-enhanced, cavitation-controlled and cavitation-monitored HIFU therapy has thus been developed, which enables suc- cessful tissue ablation at acoustic energies lower than in current clinical use.

Given the great promise of High Intensity Focused Ultrasound (HIFU) as a therapeutic modality, the aim of the present study is to develop and optimise a technique that uses externally applied focused ultrasound energy and remote, ultrasound-based treatment monitoring to destroy subcutaneous fat safely, effectively and non-invasively. Based on initial cavitation and temperature measurements performed ex vivo in excised porcine fat at four different frequencies (0.5, 1.1, 1.6 & 3.4MHz) over a range of pressure amplitudes and exposure durations, it was concluded that 0.5MHz is the optimal frequency for this application since it is capable of instigating inertial cavitation at relatively modest pressures while enhancing focal heat deposition. Histological assessment of tissue treated above the cavitation threshold at 0.5MHz both ex vivo and in vivo demonstrated damage to adipocytes and connective tissue. Furthermore, a good correlation was identified between the energy of broadband emissions detected by the passive cavitation detector (PCD) and the focal temperature rise at 0.5MHz during ex vivo experimentation, which could be exploited as a tool for non-invasive monitoring of successful treatment delivery. In addition, localisation of cavitation activity by means of passive cavitation detection was achieved and shown to provide a strong indicator of the location of induced histological damage. Based on the specific requirements identified during initial experimentation, an application-specific HIFU transducer, cavitation detector and real-time treatment monitoring software was developed and tested ex vivo. This treatment system was found capable of producing extensive damage to adipocytes and collagen confined to the subcutaneous fat layer at the desired treatment depth, which coincided with the location of cavitation activity as displayed by the real-time treatment monitoring software.

High Intensity Focussed Ultrasound (HIFU) ablation is a promising technology for the non-invasive, targeted treatment of certain types of cancer. The technique functions by subjecting tumours to a cytotoxic level of intense, localised heating, while leaving the surrounding tissue unharmed. However, a number of limitations in the available HIFU treatment monitoring methods are currently hampering the effectiveness and clinical adoption of the therapy.This work aims to develop improved metrics of HIFU-induced biological damage that are specifically suited to monitoring and controlling HIFU ablation. Firstly, an optical method that enables straightforward quantification of thermal damage in protein-embedding hydrogels is developed. Secondly, hydrogels embedded with different cell lines are used to assess the performance of common temperature-based metrics of cell death across a range of HIFU-relevant conditions. Finally, a novel, passive acoustic detector designed for the real-time monitoring of HIFU-induced tissue damage is proposed. The detector is shown to predict lesioning with over 80% accuracy in regimes that are very likely to create lesions (60 J of acoustic energy or more), with an error rate of less than 6% for exposures that are too short to cause lesioning (up to 1 s long). The proposed detector could therefore provide a low-cost means of effectively monitoring clinical HIFU treatments passively and in real time.

Systemic administration of adenovirus type 5 (Ad5) vectors for the treatment of cancer is limited by poor circulation kinetics and inefficient uptake from the bloodstream into tumours. This study reports a novel method for linkage of highly-PEGylated gold nanoparticles (AuPEG) to Ad5 by a single reduction cleavable bond. The resulting ‘dandelion’ structure provides very effective steric shielding with only minimal and reversible modification of the Ad5 capsid. This ablates in vitro cell infection, improves protection against the binding of antibodies, and enhances in vivo circulation kinetics. Focused ultrasound is a promising technology for the non-invasive, targeted treatment of cancer. In the context of drug delivery, cavitational energy generated upon exposure of ultrasound contrast agents to focused ultrasound can be used as a powerful stimulus to move therapeutics over distances of hundreds of microns away from blood vessels. In addition to providing a platform for effective stealthing, conjugation of AuPEG to Ad5 also increases the effective density of Ad5. This increase in density imparts a second major advantage on the strategy, observed for the first time in the present study: denser particles are transported significantly farther by cavitation-induced microstreaming than identically-sized particles of lower density. Specifically, in in vitro tests using a tumour-mimicking flow-channel phantom model and in in vivo experiments using tumour bearing mice, Ad5–AuPEG was delivered farther from vessels in response to ultrasound induced cavitation than either naked Ad5 or polymer-coated Ad5. The enhancements in stealthing and improvements in response to ultrasound provided by this strategy enabled up to 12% (S.D. 0.97) of the injected dose to be deposited in the tumour, compared to just 0.12% (S.D. 0.05) for Ad5 without ultrasound (p < 0.001). Consequently, in a survival study, mice treated with Ad5–AuPEG with focussed ultrasound had the slowest tumour growth and longest survival rate when compared to mice treated with Ad5 alone, Ad5–AuPEG alone, or Ad5 with focussed ultrasound. These results provide compelling evidence that the combination of focussed ultrasound with density-augmented stealthed Ad5 results in improved delivery to tumours and therapeutic efficacy. This combination of ultrasound with particle modification for optimal cavitation-mediated delivery has the potential to be applied to a broad range of anti-cancer nano-medicines and therapeutics to augment their bio-availability for improved cancer treatment.

Cancer therapy is severely hampered by the poor delivery of agents out of the blood vessels and into tumours. This is due to the irregular vasculature, high interstitial pressure and dense extracellular matrix associated with tumour tissue. As a consequence, high doses of agents must be administered intravenously for effective accumulation in the tumour. This leads to a low therapeutic index, necessitates multiple administrations of the same drug, and for many cancer drugs, also leads to toxic side-effects. Increasingly complex therapeutics, such as antibodies and viruses, only exacerbate this delivery problem as their greater size leads to lower coefficients of diffusion and, consequently, even greater portions of the tumour remain untreated. There have been studies aimed at improving therapeutic outcomes using microbubble-nucleated, ultrasound-induced cavitation, which provides a mechanical impetus to drive drugs out of the vasculature and into tumours. However, microbubbles are limited by their large size, their instability in the blood and their destruction upon cavitation. This thesis details the formulation of two alternative cavitation nuclei to overcome the limitations of microbubbles: mesoporous carbon particles and polymer cups. These are solid, submicron particles that contain crevices into which nanobubbles can be stabilised. Initial studies of their biocompatibilty have indicated that these formulations may be safe for intravenous administration. A tumour mimicking phantom was first used to quantify drug delivery caused by cavitation. Both polymer cups and mesoporous carbon particles were found to significantly enhance delivery of a model therapeutic agent by this method. In vivo the polymer cups were used to enhance the delivery of an oncolytic vaccinia virus: intravenous administration of 1x105 pfu vaccinia virus, polymer cups and ultrasound treatment was shown to cause a 780-fold increase in genome copies in the tumours of a SKOV-3 tumour model, and 5,700-fold, in the tumours of a HEPG2 tumour model 20 days after the treatment. In mice treated with 1x106 pfu of virus cavitation caused by cups and ultrasound was shown to cause regression in 7 of the 8 tumours in comparison to just 1 of the 8 tumours that were treated with virus alone.