Paul Rademeyer

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.

Magnetic targeting of microbubbles against physiologically relevant flow conditions

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. 104 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.

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 ~6 cm s-1 and shear rates of ~60 s -1.

The influence of blood on targeted microbubbles

The ability to successfully target the delivery of drugs and other therapeutic molecules has been a key goal of biomedical research for many decades. Despite highly promising in vitro results, however, successful translation of targeted drug delivery into clinical use has been extremely limited. This study investigates the significance of the characteristics of whole blood, which are rarely accounted for in vitro assays, as a possible explanation for the poor correlation between in vitro and in vivo experiments. It is shown using two separate model systems employing either biochemical or magnetic targeting that blood causes a substantial reduction in targeting efficiency relative to saline under the same flow conditions. This finding has important implications for the design of targeted drug delivery systems and the assays used in their development.

Correction to 'Magnetic targeting of microbubbles against physiologically relevant flow conditions'.

[This corrects the article DOI: 10.1098/rsfs.2015.0001.].

Magnetic targeting of microbubbles at physiologically relevant flow rates

The localization of microbubbles to a target site has been shown to be essential to their effectiveness in ultrasound mediated drug delivery and gene therapy. The incorporation of super paramagnetic nanoparticles into the microbubble coating enables them to be manipulated using an externally applied magnetic field. Magnetic microbubbles have been shown to be effective in therapeutic delivery both in vitro and in vivo in a mouse model. The aim of this experiment was to determine under what conditions in the human body magnetic microbubbles can be successfully imaged and targeted. Different flow rates and shear rates were generated in a tissue mimicking phantom and targeting was observed using a 9.4 MHz ultrasound imaging probe. For the highest shear rates, targeting was also observed optically. Results indicate that magnetic microbubbles can be successfully targeted at shear rates found in the human capillary system (>1000/s) and at flow rates found in the veins and smaller arteries (~200 ml/s). Successful...

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 (<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.

High-throughput production of microbubble contrast agents using a sonofluidic device

The response of microbubbles to a given sound field is determined by their size and coating. These, in turn, depend on their chemical formulation and the production technique. Sonication is the most commonly employed method and can generate high concentrations of microbubbles rapidly but with a broad size distribution and poor reproducibility. Microfluidic devices provide excellent control over size, but the small-scale architectures required are often challenging to manufacture, offer low production rates, and are prone to clogging. Microbubbles may also have inferior surface characteristics and stability compared to those produced by sonication. In this study we investigate a hybrid technique in which monodisperse microbubbles of ~200μm diameter are produced at high flow rates in a simple T-junction and then undergo controlled fragmentation by exposure to ultrasound via an integrated transducer operating between 71-73kHz. Microbubbles were prepared using the device or a standard sonication protocol and ...

High throughput acoustic and optical characterization of microbubbles for optimized contrast ultrasound imaging

Echogenic particles, such as microbubbles and volatile liquid micro-/nanodroplets, 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 optimization of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterization 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 with a throughput of 20 samples/second and an uncertainty below 7% in the measurements. Demonstration of optimized contrast ultraso...

Investigating the effect of fabrication method on the stability and acoustic response of microbubble agents

Microbubbles stabilized by a surfactant or polymer coating are already in clinical use as ultrasound imaging contrast agents. They have also been widely investigated as vehicles for drug delivery and gene therapy that can be tracked and triggered using ultrasound. Extensive studies have been made of the effects of the coating material and gas core on microbubble characteristics, but the influence of the fabrication method has received less attention. The aim of this study was to compare the behavior of microbubbles prepared using different techniques. Phospholipid-coated microbubbles were produced using sonication, electrospraying, or in a specially designed microfluidic device. The microbubbles were observed using optical, electron, and fluorescence lifetime imaging microscopy (FLIM) to interrogate their surface microstructure and stability over time. Their acoustic response was then determined in a flow chamber by detecting the pressure scattered from individual microbubbles as they passed through the f...