Microfluidic system for high throughput characterisation of echogenic particles

Echogenic particles, such as microbubbles and volatile liquid micro/nano droplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimisation of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterisation techniques, such as ultra-high speed microscopy provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation. A co-axial flow focusing device is used to direct a continuous stream of unconstrained particles through the combined focal region of an ultrasound transducer and a laser. Both the optical and acoustic scatter from individual particles are then simultaneously recorded. Calibration of the device and example results for different types of echogenic particle are presented, demonstrating a high throughput of up to 20 particles per second and the ability to resolve changes in particle radius down to 0.1 ?m with an uncertainty of less than 3%

A new technique for microbubble characterisation and the implications to contrast enhanced ultrasound

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 (&gt;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.</p

A new technique for microbubble characterisation and the implications to contrast enhanced ultrasound

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 â 4.5 MHz), peak negative pressure (6 kPa - 400 kPa) and pulse length (3 cycles â 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.</p

Understanding the Structure and Mechanism of Formation of a New Magnetic Microbubble Formulation

Magnetic nanoparticles and ultrasound contrast agents have both been used as vehicles for therapeutic delivery. More recently, magnetic microbubbles have been developed as a new theranostic agent which combines the advantages of the individual carriers and overcomes many of their limitations. In a previous study of gene delivery using magnetic microbubbles, it was found that a combination of magnetic liquid droplets and non-magnetic phospholipid microbubbles produced higher transfection rates than magnetic microbubbles. The reasons for this were not fully understood, however. The aim of this study was to investigate the hypothesis that conjugation between the droplets and the microbubbles occurred. A combination of optical and fluorescence microscopy and ultrasound imaging studies in a flow phantom were performed. No interaction between magnetic droplets and microbubbles was observed under optical microscopy but the results from the fluorescence and acoustic imaging indicated that magnetic droplets and microbubbles do indeed combine to form a new magnetically and acoustically responsive particle. Theoretical calculations indicate that the driving force of the interaction is the relative surface energy and thus thermodynamic stability of the microbubbles and the droplets. The new particles were resistant to centrifugation, of comparable echogenicity to conventional ultrasound contrast agents and could be retained by a magnetic field (0.2T) in a flow phantom at centre line velocities of &#126;6 cm s-1 and shear rates of &#126;60 s -1.</p

Characterisation of functionalised microbubbles for ultrasound imaging and therapy

Functionalised microbubbles have shown considerable potential both as contrast agents for ultrasound imaging and as a means of enhancing ultrasound mediated therapy. With the development of advanced techniques such as quantitative ultrasound imaging and targeted drug delivery, the accurate prediction of their response to ultrasound excitation is becoming increasingly important. Characterising microbubble behavior represents a considerable technical challenge on account of their small size (&lt;10 µm diameter) and the ultrasound frequencies used to drive them in clinical applications (typically between 0.5 and 20 MHz). This chapter examines the three main techniques used for the characterization of microbubble dynamics: ultra-high speed video microscopy, laser scattering and acoustic attenuation and back scattering measurements. The principles of the techniques are introduced with examples of their applications and their relative advantages and disadvantages are then discussed. In the second half of the chapter magnetically functionalized microbubbles are used as a case study and results obtained using each of the three techniques are presented and compared. The chapter concludes with recommendations for combining different methods for microbubble characterization

Spectral imaging for microbubble characterisation

Microbubbles stabilised by an outer lipid shell have been studied extensively for both diagnostic and therapeutic applications. The shell composition can significantly influence microbubble behaviour, but performing quantitative measurements of shell properties is challenging. The aim of this study was to investigate the use of spectral imaging to characterise the surface properties of a range of microbubble formulations representing both commercial and research agents. A lipophilic dye, C-Laurdan, whose fluorescence emission varies according to the properties of the local environment, was used to compare the degree and uniformity of lipid order in the microbubble shell and these measurements were compared with the acoustic response and stability of the different formulations. The method was found to be suitable for performing rapid and hence relatively high throughput measurements of microbubble surface properties. Interestingly, despite significant differences in lipid molecule size and charge, all of the different formulations exhibited highly ordered lipid shells. Measurements of liposomes with the same composition and the debris generated by destroying lipid microbubbles with ultrasound showed that these exhibited a lower and more varied lipid order than intact microbubbles. This suggests that the high lipid order of microbubbles is due primarily to compression of the shell as a result of surface tension and is only minimally affected by composition. This also explains the similarity in acoustic response between the formulations since microbubble dynamics are determined by the diameter and shell viscoelastic properties that are themselves a function of lipid order. Within each population, there was considerable variability in lipid order and response between individual microbubbles, suggesting the need for improved manufacturing techniques. In addition, the difference in lipid order between the shell and lipid debris may be important for therapeutic applications in which shedding of shell material is exploited e.g. for drug delivery

Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a "molecular rotor" embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component