This thesis compares theoretical models of ultrasound contrast agents to the acoustic response from single Microbubbles(MBs). The acoustic response was compared using a range of driving parameters. A rigid shelled contrast agent and a lipid shelled contrast agent were used in the comparison. While attempts to model the behaviour of some contrast agents at low mechanical index (MI) have been successful at higher MI the behaviour of MBs is still not well understood. Understanding and predicting the response ofMBs to medical ultrasound can lead to improvements in the clinical use of MBs through improved contrast agent design or improved signal processing. Numerical models were developed to compare to three specific cases; 1) Rigid shelled contrast agents 2) Lipid shelled contrast agents 3) Responses from lipid shelled contrast agents that are hit by subsequent driving pulses. Three models were used to compare to the responses from single rigid shelled contrast agents. Two of these models have been used before and the third was developed based on the optical observations of the responses of these rigid shelled agents at these MI. Two shelled models were used to compare to the response of single lipid shelled MBs. Using statistical methods the parameters defining the shell properties were found. The parameters that gave best agreement with the lipid shelled data was then used with a model to account for the molecular diffusion of gas from a MB and a new model to account for the optically observed shedding of the shell from a MB to compare to the multiple response from single MBs. While the theoretical prediction of an acoustic response of a suspension of MBs or the radial oscillation of single MBs has been compared before to experimental data, the successful comparison of the acoustic response of single MBs to the theoretical prediction is the first of it’s kind known to the author. The new theoretical model of the rigid shelled MB that was developed in this thesis gave better agreement with the experimental data than the other previously used models. The shell parameters of the lipid shelled MB were determined for the lowest driving amplitude and were in agreement with those measured previously from optical observations. Finally, the model for the shedding of the shell was shown to give quantitative agreement with the multiple acoustic responses from single MBs. When shedding of the shell was included the choice of constitutive equation for the shell was shown to strongly affect subsequent responses from the MB.

Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19973号 / 薬科博第64号 / 新制||薬科||7(附属図書館) / 33069 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The utility of microbubble agents in a variety of diagnostic and therapeutic ultrasound techniques has been widely demonstrated, most notably in Contrast Enhanced Ultrasound (CEUS) imaging. Unfortunately, the underlying mechanisms of their response to ultrasound excitation are poorly understood, restricting the development of promising techniques, such as quantitative perfusion imaging. A significant reason for this is that current microbubble characterisation techniques suffer from one or more of the following limitations: i) large experimental uncertainties, ii) physical restrictions on microbubble response and iii) failure to provide large data sets suitable for statistical analysis. This thesis presents a new technique to overcome these limitations. A co-axial microfluidic device is used to hydrodynamically confine microbubbles through the focal region of a laser and ultrasound field. The magnitude of light scattered by isolated microbubbles during ultrasound excitation is converted to radius using Mie Scattering theory. This technique is capable of obtaining large samples (>10³/min) of microbubbles to be efficiently characterised. The response of a commercial contrast agent, SonoVue®, is first investigated for a range of ultrasound exposure parameters; frequency (2 MHz - 4.5 MHz), peak negative pressure (6 kPa - 400 kPa) and pulse length (3 cycles - 8 cycles). Second the device is used to investigate the effect of composition and fabrication on microbubble response to similar ultrasound conditions. The results demonstrate a very large variability in microbubble response independent of initial size, indicating a significant lack of uniformity of coating properties. This is further supported by quantitative fluorescence imaging and quasi-static pressure chamber measurements. The implications of the findings for CEUS imaging and the development of microbubble contrast agents are discussed, as well as the limitations and suggested improvements of the characterisation technique.

Ultrasound-mediated drug delivery has been proposed as a safe and non-invasive method to achieve localized drug release. Drug-loaded microbubbles are injected in the vascular system and ultrasound waves are then used to localize and burst the microbubbles at a specific targeted area. The relatively large size of microbubbles however limits both their lifetime and their reach in the human body. Phase-change liquid droplets can extend the use of ultrasound contrast agents for localized drug delivery. Their smaller size provides several advantages. The droplets can reach smaller capillaries, such as those in tumors vasculature. Their lifetime is also considerably prolonged. Through the phenomenon of Acoustic Droplet Vaporization (ADV), triggered by ultrasound stimulation, the liquid-filled droplets experience a phase change and are converted into gas-filled microbubbles. The newly created microbubbles can then be disrupted by further stimulation and release their drug load in the tumor tissue. In this project, a protocol to image and burst perfluoropentane-based micro-sized droplets using a single transducer is developed using the Verasonics Ultrasound System. The pulse sequences are developed to allow close monitoring of the drug delivery by capturing a series of images before and after the vaporization or destruction of the droplets. The droplets response was assessed for different pulse voltages and durations. Mean pixel value was calculated for the regions of interest, using the images captured before and after delivery of the ultrasound pulse. Vaporization of the droplets can be achieved with low voltage (10V), whereas high voltage (50V) triggers their destruction. Combined with high voltage, pulse duration affects the rate at which droplets can be destructed.

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The natural dynamics are not ideal or linear. To understand their complex behavior, we needs to study the nonlinear dynamics in more simple models. This thesis is consist of two main setups. Both setups are simplified models for the behavior occurs in the complex systems. We studied in both systems the same nonlinear dynamics such as higher-harmonics, sub-harmonics, solitary waves,...etc. In Chapter (2), the propagation of nonlinear waves in a lattice of repelling particles is studied theoretically and experimentally. A simple experimental setup is proposed, consisting in an array of coupled magnetic dipoles. By driving harmonically the lattice at one boundary, we excite propagating waves and demonstrate different regimes of mode conversion into higher harmonics, strongly in influenced by dispersion. The phenomenon of acoustic dilatation of the chain is also predicted and discussed. The results are compared with the theoretical predictions of FPU equation, describing a chain of masses connected by nonlinear quadratic springs. The results can be extrapolated to other systems described by this equation. We studied theoretically and experimentally the generation and propagation of kinks in the system. We excite pulses at one boundary of the system and demonstrate the existence of kinks, whose properties are in very good agreement with the theoretical predictions, that is the equation that approaches, under the conditions of our experiments, the one corresponding to full model describing a chain of masses connected by magnetic forces. The results can be extrapolated to other systems described by this equation. Also, In the case of a lattice of finite length, where standing waves are formed, we report the observation of subharmonics of the driving wave. In chapter (3), we studied the propagation of intense acoustic waves in a multilayer crystal. The medium consists in a structured fluid, formed by a periodic array of fluid layerswith alternating linear acoustic properties and quadratic nonlinearity coefficient. We presents the results for different mathematicalmodels (NonlinearWave Equation,Westervelt Equation and Constitutive equations). We show that the interplay between strong dispersion and nonlinearity leads to new scenarios of wave propagation. The classical waveform distortion process typical of intense acoustic waves in homogeneous media can be strongly altered when nonlinearly generated harmonics lie inside or close to band gaps. This allows the possibility of engineer a medium in order to get a particular waveform. Examples of this include the design of media with effective (e.g. cubic) nonlinearities, or extremely linear media. In chapter (4), the oscillatory behavior of a microbubble is investigated through an acousto-mechanical analogy based on a ring-shaped chain of coupled pendula. Observation of parametric vibration modes of the pendula ring excited at frequencies between 1 and 5 Hz is considered. Simulations have been carried out and show spatial mode, mixing and localization phenomena. The relevance of the analogy between a microbubble and the macroscopic acousto-mechanical setup is discussed and suggested as an alternative way to investigate the complexity of microbubble dynamics. / La dinámica natural no es ideal ni lineal. Para entender su comportamiento complejo, necesitamos estudiar la dinámica no lineal en modelos más simples. Esta tesis consta de dos configuraciones principales. Ambas configuraciones son modelos simplificados de el comportamiento que se produce en los sistemas complejos. Estudiamos en ambos sistemas la misma dinámica no lineal como son la generación de armónicos superiores, los sub-armónicos, las ondas solitarias, etc. En elCapítulo (2), se estudia, tanto teórica comoexperimentalmente, la propagación de ondas no lineales en sistemas periodicos de partículas acopladas mediante fuerzas repulsivas. Se propone una configuración experimental simple, que consiste en una matriz de dipolos magnéticos acoplados. Inyectando armónicamente la señal en un extremo, excitamos ondas de propagación y demostramos diferentes regímenes de conversión de modos en armónicos, fuertemente influenciados por la dispersión. También se predice y se discute el fenómeno de dilatación acústica de la cadena. Los resultados se comparan con las predicciones teóricas de la ecuación FPU, describiendo una cadena de masas conectadas por muelles cuadráticos no lineales. Los resultados pueden ser extrapolados a otros sistemas descritos por esta ecuación. Estudiamos también teórica y experimentalmente la generación y propagación de kinks. Excitamos pulsos en la frontera del sistema y demostramos la existencia de kinks cuyas propiedades están en muy buen acuerdo con las predicciones teóricas, es decir, con la ecuación que aproxima bajo las condiciones de nuestros experimentos la correspondiente al modelo completo que describe un cadena de masas conectadas por fuerzas magnéticas. Los resultados pueden ser extrapolados a otros sistemas descritos por esta ecuación. Además, en el caso de una red finita, donde se forman ondas estacionarias, describimos la observación de subarmónicos del armónico principal. En el capítulo (3), estudiamos la propagación de ondas acústicas intensas en un cristal multicapa. El medio consiste en un fluido estructurado, formado por un conjunto periódico de capas fluidas con propiedades acústicas lineales alternas y coeficiente de no linealidad cuadrática. Presentamos los resultados de diferentes modelos matemáticos (ecuación de ondas no lineal, ecuación de Westervelt y ecuaciones constitutivas). Mostramos que la interacción entre la fuerte dispersión y la no linealidad conduce a nuevos escenarios de propagaciónde ondas. El proceso de distorsión de la onda clásica, típico de las ondas acústicas intensas en medios homogéneos, puede ser alterado de forma importante cuando los armónicos generados no linealmente se encuentran dentro o cerca del gap. Esto permite la posibilidad de diseñar un medio con el fin de obtener una forma de onda en particular. Ejemplos de esto incluyen el diseño demedios con no linealidad efectiva (por ejemplo, cúbica) o medios extremadamente lineales. En el capítulo (4), el comportamiento oscilatorio de una microburbuja se investiga a través de una analogía acusto-mecánica basada en una cadena en forma de anillo de péndulos acoplados. Se estudian los modos de vibración paramétrica del anillo pendular excitado a frecuencias entre 1 y 5 Hz. Se han llevado a cabo simulaciones que muestran la presencia de modos espaciales, mixtos y fenómenos de localización. Se discute la relevancia de la analogía entre una microburbuja y la configuración macroscópica acústico-mecánica y se sugiere como una vía alternativa para investigar la complejidad de la dinàmica de microburbujas. / La dinàmica natural no és ideal ni tampoc lineal. Per entendre el seu comportament complex, es necessita estudiar la dinàmica no lineal dels models més simples. Aquesta tesi consisteix en l'estudi de dues configuracions principals, que són models simplificats del comportament que es produeix en els sistemes complexos. Estudiem en ambdós sistemes la mateixa dinàmica no lineal, com és la generació d'harmònics superiors, sub-harmònics, ones solitàries, etc. En el capítol (2), estudiem, tant teòrica com experimentalment, la propagació de les ones no lineals en sistemes periòdics de partícules acoblades mitjançant forces repulsives. Es proposa una configuració experimental simple, que consisteixen en una matriu de dipols magnètics acoblats. En conduint harmònicament la xarxa en un límit, excitemla propagació de les ones i demostrem diferents règims de conversió de modes en harmònics més alts, força influenciada per la dispersió. El fenomen de la dilatació acústica de la cadena també es prediu i es discuteix. Els resultats es comparen amb les prediccions teòriques que descriu una cadena de masses conectades per molls quadràtics no lineals. Els resultats es poden extrapolar a altres sistemes descrits per aquesta equació. Hem estudiat teòrica i experimentalment la generació i propagació de Kinks. Excitem polsos a la frontera del sistema i demostrem l'existència d'Kinks, les propietats desl quals estan en molt bon acord amb les prediccions teòriques, és a dir, de l'equació que aproxima sota les condicions dels nostres experiments la corresponent al model complet que descriu un cadena demasses connectades per forcesmagnètiques. Els resultats es poden extrapolar a altres sistemes descrits per aquesta equació. A més, en el cas d'una xarxa finita, on es formen ones estacionàries, descrivim l'observació de subarmónicos de l'harmònic principal. En el capítol (3), s'estudia la propagació d'ones acústiques intenses en un medi multicapa. El medi consisteix en un fluid estructurat, format per una matriu periòdica de capes de fluid amb l'alternança de propietats acústiques lineals i coeficient de no linealitat de segon grau. Es presenten els resultats per a diferents models matemàtics no lineals (equació d'ones no lineal, equació de Westervelt i les equacions constitutives). Es demostra que la interacciò entre la forta dispersió i no linealitat condueix a nous escenaris de propagació de l'ona. El procés de distorsió en formad'ona clàssica, típica de les ones acústiques intenses en medis homogenis, es pot alterar de manera significativa quan els harmònics generats de forma no lineal es troben dins o a prop del gap. Això obri la possibilitat de dissenyar unmedi per tal d'obtenir una forma d'ona particular. Exemples d'això inclouen el disseny delsmedis amb una no linealitat efectiva (per exemple cúbica), o medis extremadament lineals. En el capítol (4), el comportament oscilatori d'una micro-bombolla és investigat a través d' una analogia acústica-mecànica basada en una cadena en forma d'anell de pèndols acoblats. Es considera l'observació dels modes de vibració paramètriques de l'anell pendular excitat amb freqüències entre 1 i 5 Hz. S'han dut a terme simulacions que mostren la presència de moes espacilas, mixtes i fenòmens de localització. Es discuteix la relevància de l'analogia entre les microbambolles i la configuració macroscòpica acústica-mecànica i es suggereix una formaalternativa per a investigar la complexitat de la dinàmica demicrobombolles. / Mehrem Issa Mohamed Mehrem, A. (2017). Nonlinear acoustics in periodic media: from fundamental effects to applications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/80289 / TESIS

Ultrasound has been established as one of the most widely used imaging modalities for diagnostic purposes, due to the several advantages it provides in comparison with other techniques. Hence, ways to further improve the confidence in diagnoses provided by ultrasound are constantly being investigated. One of them is the introduction of Ultrasound Contrast Agents, which can enhance the weak echoes produced by the small vessels, improving the imaging performance. In this study, a setup was created and six ultrasound imaging techniques were implemented by using the Verasonics Research System®, in order to take advantage of the different behavior between the tissue and the Polyvinyl-Alcohol microbubbles, when exposed to ultrasound. These were: Fundamental B-mode, Ultraharmonic, PulseInversion, Subharmonic Pulse Inversion, Ultraharmonic Pulse Inversion, Combination of the Sub- and Ultraharmonic Pulse Inversion. For the assessment of the bubbles’ response, the amplitude spectra were used, which showed a limited detection around the ultraharmonic region. For the evaluation of the imaging performance of the techniques, the Contrast-to-Tissue (CTR) and Contrast-to-Noise Ratios (CNR) were calculated. The Combination of the Sub- and Ultraharmonic Pulse Inversion reported the highest imaging performance among all the techniques. A comparison with previous articles provided a similar pattern in terms of CTR. / Technology

The current methodologies in body scanning diagnostic uses different simultaneous imaging modalities like Ultrasound (US), magnetic resonance imaging (MRI), single photon emission tomography (SPECT) and positron emission tomography (PET). The field requires combination of different modalities for effective use in clinical diagnostics. Such incorporation of different modalities has already been achieved. For example, PET-CT hybrid scanner is designed to acquire align functional and anatomical images and recently US-MRI scanner has successfully shown to improve diagnosis of prostate cancer. The non ionizing radiation hybrid US-MRI is of great interest in health care industry. Further these US and MRI modalities uses different contrast agents like micro-sized gas bubbles (MBs) encapsulated by surfactant for US and superparamagnetic nanoparticles for MRI imaging modalities to further enables new diagnostic opportunities and therapeutic applications. Recently in our 3MiCRON project, we have developed the multimodal contrast agent that could be supported for both US and MRI. This was achieved by coating the magnetic nanoparticles to the poly vinyl alcohol (PVA) surfactant shelled MBs. The nanoparticles in the shell effect the structure can alter the MBs performance as an ultrasound contrast agent. The present thesis is conducted to examine the acoustic and mechanical properties of such multimodal contrast agents. These multimodal contrast agents were prepared by coating the surface of PVA-shelled MBs by two following strategies: (1) The superparamagnetic iron oxide (Fe3O4) nano-particles (SPIONs) were chemically anchored to the surface of poly vinyl alcohol (PVA) shelled MBs namely MBs-chem and (2) in the second strategy the SPIONs were physical entrapped into the PVA shell while formation of PVA surface on the gas bubble were named as MBs-phys. To understand the scattering efficiency and viscoelastic properties of these modified agents, we investigated the backscattering power, attenuation coefficient and phase velocity measurements. Our acoustic experimental results indicate that both the modified MBs and non-modified plain PVA-shelled ultrasound contrast agents have the same echogenic response. The investigation of mechanical properties of modified MBs revealed that the attached SPIONs on the PVA shell has reduced the stiffness of MBs-chem shell, while, the SPIONs inside the shell has increased MBs-phys stiffness. As a result, MBs-chem exhibits soft shell behavior under ultrasound exposure than both MBs-phys. Finally, the images were obtained through the MRI investigations at the department of Radiology, Karolinksa Institute, has demonstrated that both MB types have enough magnetic susceptibility that further provides good detectability in vitro and in vivo. As an outlook, the modified magnetic gas bubbles, i.e. both MBs-chem and MBs-phys can be proposed as a potential contrast agent for both US and MR imaging and can be further utilized in potential therapeutic applications. / <p>QC 20131126</p>