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
