Wall influence on dynamics of a microbubble

The nonlinear dynamic behaviour of microscopic bubbles near a wall is investigated. The Keller-Miksis-Parlitz equation is adopted, but modified to account for the presence of the wall. This base model describes the time evolution of the bubble surface, which is assumed to remain spherical, and accounts for the effect of acoustic radiation losses owing to liquid compressibility in the momentum conservation. Two situations are considered: the base case of an isolated bubble in an unbounded medium; and a bubble near a solid wall. In the latter case, the wall influence is modeled by including a symmetrically oscillating image bubble. The bubble dynamics is traced using a numerical solution of the model equation. Subsequently, Floquet theory is used to accurately detect the bifurcation point where bubble oscillations stop following the driving ultrasound frequency and undergo period-changing bifurcations. Of particular interest is the detection of the subcritical period tripling and quadrupling transition. The parametric bifurcation maps are obtained as functions of non-dimensional parameters representing the bubble radius, the frequency and pressure amplitude of the driving ultrasound field and the distance from the wall. It is shown that the presence of the wall generally stabilises the bubble dynamics, so that much larger values of the pressure amplitude are needed to generate nonlinear responses.Comment: 25 pages, 14 figure

Phase change events of volatile liquid perfluorocarbon contrast agents produce unique acoustic signatures

Phase-change contrast agents (PCCAs) provide a dynamic platform to approach problems in medical ultrasound (US). Upon US-mediated activation, the liquid core vaporizes and expands to produce a gas bubble ideal for US imaging and therapy. In this study, we demonstrate through high-speed video microscopy and US interrogation that PCCAs composed of highly volatile perfluorocarbons (PFCs) exhibit unique acoustic behavior that can be detected and differentiated from standard microbubble contrast agents. Experimental results show that when activated with short pulses PCCAs will over-expand and undergo unforced radial oscillation while settling to a final bubble diameter. The size-dependent oscillation phenomenon generates a unique acoustic signal that can be passively detected in both time and frequency domain using confocal piston transducers with an ‘activate high’ (8 MHz, 2 cycles), ‘listen low’ (1 MHz) scheme. Results show that the magnitude of the acoustic ‘signature’ increases as PFC boiling point decreases. By using a band-limited spectral processing technique, the droplet signals can be isolated from controls and used to build experimental relationships between concentration and vaporization pressure. The techniques shown here may be useful for physical studies as well as development of droplet-specific imaging techniques

Non Linear Ultrasound Doppler and the Detection of Targeted Contrast Agents

One of the main challenges in molecular imaging with targeted contrast agents is the detection and discrimination of attached agents from the rest of the signals originating from freely flowing agents and tissue. The aim of this thesis was to develop methods for the detection of targeted microbubbles. One approach consisted of investigating the use of nonlinear Doppler for this purpose. Nonlinear Doppler enables the differentiation of moving from non-moving and linear from nonlinear scattering. Targeted microbubbles are static and nonlinear scatterers and they should be detected using this technique. A novel nonlinear Doppler technique: Pulse subtraction Doppler, was developed and compared to pulse inversion Doppler. It is shown that both techniques lead to similar Doppler spectra and depending on the medical applications and the equipment limitations, both techniques have benefits. This served as a starting point for the derivation of a generalised nonlinear Doppler technique, based on combined linear pulse pair sequences and tested in a simulation study. The response from a single microbubble was simulated for different pulse combinations and the pulse sequences were compared with regards to criteria specific to imaging requirements. It was shown that depending on initially set criteria, such as transmitted energy, mechanical index or scanner characteristics, certain pulse combinations offer alternatives to the current imaging modalities and allow to take into account specific constrains due to the targeted application/equipment. Furthermore, the proposed approach is directly applicable in a strict non linear imaging approach, without Doppler processing. An in vitro phantom was designed in order to assess pulse subtraction Doppler for the detection and discrimination of static nonlinear microbubbles in the presence of free flowing ones. It was shown that pulse subtraction Doppler enables such discrimination and the practicability for in vivo situations is discussed. The pulse subtraction Doppler sequences were also tested on a phantom containing magnetic bubbles. It was shown that the magnetic bubbles can be immobilised through a magnetic field to a specific region of interest under flow conditions. The bubbles also showed to be acoustically detectable and to scatter linearly at diagnostic driving pressures. Preliminary work regarding experimental biotinylated microbubbles and their attachment to streptavidin coated surfaces is also presented. Due to their proximity to a wall, researchers have found that targeted microbubbles exhibit different acoustic signatures compared to free ones and this knowledge can improve their detection techniques. The behaviour of microbubbles against a membrane of varying stiffness was also studied through high speed camera observations. It was found both experimentally and by comparison to theoretical modelling that within the stiffness range of human blood vessels the change in acoustical behaviour of microbubbles is negligible. This thesis has taken two complementary research approaches which have shown to constitute advancements for the detection and discrimination of targeted microbubbles

Molecular mechanisms of sonoporation in cancer therapy : Optimization of sonoporation parameters and investigations of intracellular signalling

Background: Sonoporation, which is treatment with ultrasound (US) and microbubbles (MB), has shown great potential for enhancing the therapeutic efficacy of chemotherapy in cancer therapy. However, there is still very little consensus regarding the mechanism or optimal experimental and therapeutic parameters. The original assumption was that pore formation in the cell membrane was responsible for the increased uptake of drugs, but it is currently understood that the mechanisms are far more complex. The field combines US physics, MB formulation and physics, (cell) biology, pharmacology, pharmacokinetics and the biodistribution of both drugs and MBs. Hence, there is an almost endless range of experimental parameters and potential bioeffects. The current literature includes a plethora of experimental setups and parameters, which complicates the clinical translation of sonoporation. Aims and methodology: In this thesis, the effects of low-intensity US and MB parameters were investigated in vitro using custom-made ultrasound chambers and correlating commonly used measures as uptakes of impermeable dye (i.e. flow cytometry) and viability to detect intracellular signalling responses to sonoporation in different cell types. Intracellular signalling responses to sonoporation are largely unknown, and their influence on key proteins in important signalling pathways have been elucidated using phosphoflow cytometry. To gain the understanding and translatability of US + MB parameters, three commercially available MB formulations were characterized, and important parameters, such as dose and formulation, were investigated in vitro and the in vivo enhancement of chemotherapy in a mural model of pancreatic ductal adenocarcinoma (PDAC). Results and conclusions: Effective sonoporation was achieved using commercial microbubbles and low-intensity US in the diagnostic range, both in vitro and in vivo. In the low-intensity US regimen, effective sonoporation required MBs, and the efficacy increased as US intensity and MB concentrations increased. The choice of optimal MBs depended on the US parameters used, and must be carefully chosen based on the therapeutic context. The findings in vivo were correlated to those in the in vitro experiments and to simulations on MB behaviour. Sonoporation induced the immediate, transient activation of intracellular signalling (MAPK-kinases; p38, ERK1/2, CREB, STAT3, Akt) as well as changes in the phosphorylation status of the proteins involved in protein translation (i.e. ribosomal protein S6, 4E-BP1 and eIF2α). The intracellular signalling response resembles cellular recovery after pore formation by electroporation and pore-forming toxins. Based on this observation, we hypothesize that sonoporation induces a cellular stress response that is related to the membrane repair and restoration of cellular homeostasis, and it may be exploited therapeutically. Varying responses in different cell types better represent the variability within a tumour, and they indicate that the effects on the tumour microenvironment may be important for sonoporation efficacy. In the present work, cellular stress was induced using low-intensity US below the intensity limit approved for diagnostic imaging, and healthy blood peripheral cells were minimally affected

Optimizing the performance of phase-change contrast agents for medical ultrasonography

In the past two decades, perfluorocarbon (PFC) droplets have been investigated for biomedical applications in several imaging modalities. More recently, interest has increased in 'phase-change' PFC droplets (or 'phase-change' contrast agents (PCCAs)), which can convert from liquid to gas with an external energy input. In the field of ultrasound, phase-change droplets present an attractive alternative to traditional microbubble agents for many diagnostic and therapeutic applications. In this thesis, new techniques are presented to enhance the performance of PCCAs and ultimately drive the platform closer to clinical use. It is demonstrated that the efficiency of activation can be improved by incorporating highly volatile compounds, and new particle generation methods are developed to produce PCCAs from these compounds. Next it is shown that these methods can be adapted to highly tune the performance of the droplets with regard to both sensitivity to ultrasound and thermal stability - allowing one to 'tune' the droplets for an intended application. Next, an alternative method of determining appropriate ultrasound activation parameters for nanoscale emulsions is demonstrated based on changes in the bubble population produced. Through high-speed video microscopy,the physics of particle expansion after droplet activation are studied to show that droplets produce unique, size-dependent acoustic 'signatures' during vaporization that can be detected at diagnostic ultrasound frequencies and used to isolate droplet vaporization events from tissue and standard microbubble contrast agents. Finally, the benefits of these techniques both in vitro and in vivo are demonstrated in applications such as ultrasound diagnostic and molecular imaging, ultrasound-mediated tissue ablation, and drug delivery across the blood-brain barrier. The results shown here demonstrate that PCCAs can be highly tuned to perform ideally across a wide range of ultrasound-related applications, which bolsters the argument for their use as diagnostic and therapeutic clinical ultrasound agents. Future refinement of the techniques in this thesis will help drive PCCAs toward eventual use in improving human health.Doctor of Philosoph

Characterisation of adherent microbubbles for molecular targeted ultrasound

Molecular imaging is a field of medicine which can offer great potential for both diagnostic and therapeutic purposes. Within this field contrast enhanced ultrasound displays the possibility of making molecular imaging a cost effective viable tool in an increasingly diverse set of clinical situations. One of the current challenges associated with this technique is how one differentiates the signal for adherent microbubbles from those produced by the bulk non-adherent population. The first part of this thesis acoustically examines the response of single microbubbles under the effects of adhesion and compares the response observed with that of the MBs non-adherent counterpart. It was found experimentally that differences could be observed in both the 2nd harmonic signals generation and in the stability over repeated exposure. These differences could be utilised as the basis for discretisation imaging strategies. The second section of this thesis attempts to characterize these differences in terms of current theoretical models. A more comprehensive modelling strategy is utilised for the fitting of increasingly complex theoretical models. Good agreement was found with the outputs of this fitting procedure with previously reported parameters. Further detail could also be observed in the form of various size/resonance effects which have not previously been reported. There was little observed difference between the parameters extracted for the adherent and non-adherent MBs although it was suggested that the effective elasticity of an adherent MB could be elevated in comparison to its non-adherent counterpart in the region of resonance. Efforts will be required to control and account for some of the variability observed in MB response before this can be stated definitively however.Open Acces

Microbubbling and microencapsulation by co-axial electrohydrodynamic atomization

Microbubbles coated with polymers or surfactants have been used in medical imaging for several years as ultrasound contrast agent particles and are now being investigated by researchers as drug and gene delivery vehicles and blood substitutes. Current methods available for the preparation of microbubbles are insufficient as they result in microbubbles with a wide size distribution and as such filtration is necessary before their use. With a view to fill the above demand, a detailed investigation has been carried out in this research to learn the viability of co-axial electrohydrodynamic atomization (CEHDA) technique to prepare microbubbles. The research also focuses on the effects of the process parameters such as flow rates, applied voltage and material parameters such as electrical conductivity, surface tension and viscosity with the objective of preparing polymer or surfactant coated stabilized microbubbles with diameters < 8 μm and with a narrow size distribution. A model glycerol-air system was used so that the CEHDA technique was modified to generate suspensions of microbubbles to a diameter < 8 μm with a narrow size distribution and then to characterise the CEHDA microbubbling process in terms of size and stability with varying process parameters and material parameters. Construction of a parametric plot between the air flow rate and the liquid flow rate was extremely useful in identifying the flow rate regime of air and liquid or suspension or solution for the continuous microbubbling of the system used. With further investigations into the CEHDA microbubbling technique, it was possible to develop strategies, first, to prepare suspensions of stabilized phospholipids-coated microbubbles with a mean diameter of ~ 5 μm and a polydispersivity index of 9%, and second, polymeric microspheres with a mean diameter of 400 nm and a polydispersivity index of 8% using a biocompatible polymer