Advanced characterisation study of ultrasound contrast bubbles in their natural hydrated state

This study presents a systematic investigation on in-house ultrasonic contrast agents, known as microbubbles (MBs), in their natural hydrated state. Contrast microbubbles have strong acoustic scatter profiles that significantly enhance ultrasonic visualisation of the human vasculature. Understanding and characterising the behaviour and morphological properties of these microbubbles is of interest and is the main research aims of this thesis - currently there are only a few clinically approved microbubbles. To manufacture clinically translatable theranostic vehicles, it is imperative to understand the mechanical and nanostructural properties of these bubbles in vitro; this will enrich the understanding of how their structural, biophysical and chemical properties impact their functionality in vivo. The behaviour and morphological properties of microbubbles have not been fully explored, this includes the lipid arrangement of the shell membrane. Hence, the work of this thesis is centred around exploring the physical properties of the bubbles. In particular, the microbubble shell is investigated in detail by applying complementary, state-of-the-art, experimental techniques such as atomic force microscopy and cryogenic focused-ion-beam scanning electron microscopy. The phospholipid microbubbles used throughout have been manufactured using microfluidic technology; the gaseous phase would intersect at a T-junction with the hydrophilic liquid phase, producing contrast bubbles by exploiting the amphiphilic properties of phospholipids. Subsequent ultrasound investigations have investigated their attenuation capabilities (dB cm-1 ) over the 12 - 55 MHz frequency range. The results from these studies indicated a sub-population of sub-micron bubbles due to the higher attenuation seen at higher frequencies. Then, using nanoparticle tracking analysis, resonant mass measurements and optical brightfield microscopy, data has been generated to reveal the complete size distribution of bubbles produced using the patented microfluidic method for generating ultrasound contrast bubbles. One of the key findings of this thesis was the discovery of sub-micron (<1000 nm) and micron-sized bubbles in solution. This finding then allowed for both the distributions to be investigated using atomic force microscopy and cryogenic focused ion-beam scanning electron microscopy. Atomic force microscopy (in combination with an optical microscopy set up) is used extensively to directly visualise the shell membrane and its nanostructural components. This led to the quantification of the thickness of shell membranes as well as the observation of morphological changes that occur during bubble deformation and, ultimately, their collapse. AFM imaging mode techniques such as tapping mode and quantitative imaging mode have allowed for the thickness and lipid configuration of phospholipid-shelled MBs to be quantified for the first time, a key finding in this thesis. A shell thickness of ~6.5 nm has been found using atomic force microscopy, leading to the proposal of the membranes being tri-layered; undertaking a {hydrophilic head-hydrophobic tail}-{hydrophobic tail - hydrophilic head}-{hydrophilic head hydrophobic tail} - configuration. Further work using force-curve mode AFM has also been conducted to measure mechanical forces at the nanoscale. This AFM mode generated force spectroscopy data with enough force resolution that, when combined with elastic models, gave insight into the interactions, and mechanical responses that these bubbles elicit when undergoing force compression. Bubble stiffness and Young's Modulus have been calculated using different mechanical theories to evaluate which is most appropriate for analyses on the soft matter bubble systems. In this context, it has been shown for the first time comprehensively (accounting for the now measured shell thickness) that the linear elastic Reissner Model is not a suitable one as it can overestimate (GPa range) or underestimate (KPa range) if the correct shell thickness value is not used. The high force resolution allowed for investigating the polyethylene glycol brushes end-grafted to the phospholipid microbubbles. Using the Alexander-de Gennes polymer brush theory revealed overestimated PEG- brush thickness values, which could also be affected by ionic/DVLO forces due to the curvature of the bubble shell. Cryogenic focused-ion beam scanning electron microscopy is a valuable technique for the detailed study of soft matter systems. The focused-ion-beam allowed for probing deep into the cryopreserved sample by milling through the upper surface to expose suspended bubbles. Cryo imaging through focused ion beam-scanning electron microscopy allowed for probing sub-micron contrast bubbles under conditions that are close to in vivo. This work corroborated the results highlighting a trilayer configuration of lipids (~8 nm when taking into account the PEG brush), and provided novel information on the structure of the shell membrane and heterogenic lipid domain formation, which could have implications in drug/gene loading capabilities. In conclusion, this study provides systematic characterisation of in-house phospholipid-shelled contrast bubbles using various advanced techniques to characterise mechanical, nanostructural, and size properties across the nano and microscale. This study is the first to offer such a comprehensive report on the properties of phospholipid contrast bubbles in their natural hydrated states. Thus, cutting edge techniques and improved methods for bubble imaging are presented, which can be used to propel the development of theranostic contrast bubbles.