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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.

Inertial cavitation occurring during HIFU exposure can act both as a promoter and as a marker of heat deposition, because bubbles re-radiate part of the incident field as broadband noise emissions that are detectable over a frequency range far removed from the main HIFU excitation frequency. In the present work, a commercially available ultrasound scanning system (with the transmit signal turned off) and linear array are used to detect the emissions emanating from cavitating bubbles passively on 64 independent channels during HIFU exposure. A novel algorithm is presented that enables localization and mapping of cavitation activity, both for the case of a single bubble and in the context of contiguous, disjoint cavitating regions. By contrast to B-mode hyperechogenicity imaging that is widely used in ultrasound-guided HIFU systems but can only operate whilst the HIFU is off, the passive imaging technique presented here can be implemented during HIFU exposure, thus providing a potential means of real-time treatment monitoring during ablation.

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.