Gas filled microbubbles (MBs) stabilized by a shell (e.g. lipids) are commonly used as ultrasound (US) contrast agents. Attaching biomolecules to the surface of MBs allows for molecular US imaging of various diseases. With the increased interest in targeted US imaging, new platforms to prepare disease-targeted MBs are necessary. Furthermore, attaching signaling agents to MBs creates multi-modal imaging opportunities, enhancing visualization and quantification of disease biomarkers. In this thesis, MBs labeled with 99mTc and/or rhodamine dye by taking advantage of the strong interaction between biotin and streptavidin are reported. Radiolabeling of MBs was achieved in good radiochemical yield (~ 30%). 99mTc-labeled MBs were targeted to vascular endothelial growth factor receptor 2 (VEGFR2) using an anti-VEGFR2 antibody and to prostate specific membrane antigen (PSMA) using small-molecule based PSMA inhibitors. In vitro evaluations showed successful binding of MBs to the target while in vivo targeting assessments were unsuccessful. New strategies to target MBs to the site of interest were then developed through the use of the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO). A biotinylated derivative of Tz was loaded on streptavidin coated MBs to create a Tz-derivatized MB (MBTz). Targeting MBTz to extracellular markers of cancer such as VEGFR2, PSMA and urokinase plasminogen activator receptor (uPAR) in vitro was achieved using TCO-conjugated antibodies. In vivo targeting was successful for VEGFR2 and PSMA, but not uPAR. Translating the new strategy to other US contrast agents was then investigated. Gas vesicles (GVs) produced in halobacteria were conjugated with TCO using amide-coupling chemistry. A 99mTc-labeled derivative of Tz was loaded on TCO-GVs (RCY= 59%) and their distribution assessed by SPECT/CT imaging and ex vivo tissue counting. Having established a convenient platform to conjugate molecules to GVs and MBs, future work focuses on developing a new generation of human compatible molecular US imaging probes. / Dissertation / Doctor of Philosophy (PhD)

There is currently no solution for imaging fibrin-rich adhesions following surgery, yet the condition costs healthcare providers upwards of $2 billion annually. Over the past decade the development of ultrasound contrast agents has seen an increase in commercialization of generic microbubble formulations for standard diagnostic applications such as echocardiography. To enhance diagnostic power, molecularly targeted microbubbles are formulated with the addition of a ligand to the outer shell. The microbubble formulation must be modified so that the contrast agents are stable over time and targeted with the appropriate ligand while maintaining their echogenicity relative to surrounding soft tissue. We used a dual approach to look at microbubbles optically to predict their relative signal enhancement in vivo given their size distribution and concentration. An ImageJ macro script was developed based off BubblesizerJ, a previously developed open-source program. To confirm that modified microbubbles maintain acoustic properties relative to soft tissue, an agarose phantom model was developed that allows for high throughput testing of multiple microbubble formulations. / 2026-09-03T00:00:00Z

Yes / The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable Combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery. / The work was funded by the Medical Research Council (grant number: MR/L01629X MRC Medical Bioinformatics Centre) and the EPSRC (grant number EP/P023266/1 Health Impact Partnership). EPSRC (EP/I000623/1, EP/K023845/1). Laura E. McVeigh was funded by an EPSRC PhD Studentship (EP/L504993/1).

In this thesis the possibilities of using capillary electrophoresis as a separation technique for analysis of proteins and microbubbles is presented. A complete analytical process consists of five necessary steps of which one is the actual analysis step. For this step a suitable analytical technique is needed. Capillary electrophoresis (CE) is one of the common analytical separation techniques used for analysis of a diversity of analytes, and can be both used in routine analysis and for research purposes. The reason for using CE, compared to other liquid-based separation techniques, is mainly short analysis time, high resolution, and negligible sample volumes and solvent waste. Depending on the characteristics of the analytes, and the sample matrix, different modes of CE can be used, where capillary zone electrophoresis (CZE) is the most employed one. The basic principle of CZE is separation of the analytes due to differences in total mobility, which is dependent on the charge and size of the analytes, and the electroosmotic flow (EOF). The EOF can be controlled by several parameters e.g. choice of background electrolyte (BGE), and the optimization of the parameters has been discussed throughout the thesis. To improve the properties of the BGE, an ethylammonium nitrate (EAN) water solution was used as BGE for CE analysis in Paper I. The precision of the EOF with this method was determined by adjusting the pH of the BGE, the concentration of EAN in the BGE, and the electric field. Model proteins were thereafter analysed using the optimal parameters yielding a precision sufficient for routine control. One example of the applications of CE is separation of novel contrast agents, which consist of polyvinyl alcohol microbubbles (PVA-MBs). In Paper II, a method for analysis of PVA-MBs in biological samples using CE with UV-detection was developed. It was also established that intact PVA-MBs could be distinguished from ultrasound degraded PVA-MBs in the same set-up. / <p>QC 20171012</p>

Phospholipid coated, inert gas filled microbubbles (MBs) are currently in widespread use in medical applications for the enhancement of diagnostic ultrasound images, and they are promising candidates for use in the area of targeted drug/gene delivery and uptake. As phospholipid coated MBs were developed for use with diagnostic ultrasound, their behaviour under acoustic loading is well investigated, however much less is known about their response to direct mechanical loading, which will potentially prove important as the range of uses of MBs expands. This is particularly true of the existing commercially available MB products. In this thesis, atomic force microscopy (AFM) was used to investigate the mechanical behaviour of three types of commercially produced phospholipid coated MBs, Definity®, BR14 and Sonovue®, at small deformations. Force spectroscopy was used to produce force-deformation (F-Δ) curves showing how the MBs deform under mechanical loading. Definity® MBs were deformed with tipless cantilevers at high deformations (though still less than 30% of the initial height of the MB); BR14 and Sonovue® MBs were probed with both tipless and tipped cantilevers to investigate both whole-bubble deformation and also shell indentation. BR14 was limited to low deformations; Sonovue® included both low and high deformations. The F-Δ curves were used to evaluate MB stiffness and also in combination with up to four mechanical models to predict the Young’s modulus of the MBs. The suitability of Reissner, Hertz, Elastic Membrane and De Jong theories for the prediction of the Young’s modulus of the MBs was explored. In the case of Definity® MBs no correlation between MB size and stiffness was observed; however an unexpected size dependence was observed in the Young’s modulus values, possibly due to variations in the thickness of the phospholipid shell. The membrane stretching component of elastic membrane theory was found to be the most applicable model on these MBs in this higher deformation regime. However, in this regime, gas compressibility could play a role and this is not included in the model. We studied the mechanical properties of BR14 MBs at very low deformations using ‘soft’ cantilevers. In this regime, gas compressibility should play a minimal role and there are several mechanical models which may be used. These MBs demonstrated decreasing stiffness with increasing diameter, and little variation in Young’s modulus with diameter. Hertz and De Jong theories showed more realistic Young’s modulus values (compared to other models) with little observable trend. Sonovue® MBs were used for a more comprehensive study of the small and very small deformation regimes using ‘soft’, ‘hard’ and tipped cantilevers. They showed no definitive trend in MB stiffness with MB diameter. Hertz and De Jong theory were again found to be most suitable. Analysis of curves acquired with tipped cantilevers indicated that the stiffness of a localised area of the shell membrane is similar to the overall stiffness of the MB and that the apparent Young’s modulus of the membrane according to the Hertz theory is also similar to that of the MB as a whole. Generally, considering all systems, Reissner theory was found to produce large overestimates of Young’s modulus, exceeding expected values by several orders of magnitude. Hertz and De Jong theories produced underestimates, though by a much smaller margin. Elastic membrane theory worked well and produced realistic Young’s modulus values only at relatively high deformation (the stretching term) in spite of the fact that gas compressibility is not taken into account. The suitability of the models is therefore very dependent on the deformation regime of the experiment. It seems that there is scope for better models at low deformation taking into account the soft shell of the MB and possibly its specific structure. Precise structural information of the MB shells does not exist; it is not trivial to attain and should certainly be a topic of future work with additional instrumentation.

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained. Additionally, the application of an ultrasound gel to the imaging surface may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate imaging of tissues and cells with high resolution and contrast. We have recently developed a novel method for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells using MPM. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.

Phase-change contrast agents (PCCAs) are an innovative form of imaging agent with practical applications in both the research and clinical settings. PCCAs are derived from gaseous microbubbles, which are able to act as targeted-contrast agents through conjugation of a ligand that is selective for an overexpressed receptor or biomarker in a given disease. Gaseous microbubbles can be condensed to liquid phase nanodroplets, which should be sufficiently small to extravasate into cells and/or tissues given their size and stability. Once liquid nanodroplets have internalized within a given tissue, they can be "activated" back into gaseous microbubbles with ultrasound at clinically used frequencies and energy outputs. This is purposeful as microbubbles provide much greater ultrasound reflectivity than nanodroplets. In this study, PCCAs and/or microbubbles act as a targeting agent in multiple scenarios. The projects in this study include- examination of binding and internalization of targeted PCCAs with different gaseous cores within MDA-MB-231 breast cancer cells, vaporization of liquid phase nanodroplets through application of acoustic energy via focused ultrasound (FUS), and targeting vulnerable plaque in the heart with different types of targeted microbubbles under varying shear-stresses.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

Ultrasound, applied in combination with microbubbles, has potential as a means to enhance the uptake of therapeutic agents, which could include drugs and nucleic acids, into biological cells. This process is commonly referred to as 'sonoporation', and the enhanced uptake can be caused through the incident ultrasonic pressure fi eld causing radial oscillations (cavitation) in the microbubbles, amongst other possibilities. However, the mechanisms responsible for any resultant increase in cell membrane permeability are not yet fully understood. This project focussed on achieving a more fundamental understanding of these salient processes by building on a platform of previous work within the group. One strand of the project involved a complete characterisation of the performance of a rotating mirror high speed camera (Cordin 550-62) that was previously used by our group [and others] to investigate microbubble cavitation phenomena and interactions with proximal cell membranes. Speci cally, I present herein an investigation into the image formation process with this type of camera, the essence of which stymied previous data interpretations. I demonstrate that an inherent asynchrony in the exposure of pixels within individual image frames leads to a temporal anomaly. This was achieved using low cost, flashing LED lights and resulted in the extraction of an algorithm to correct for the temporal anomaly. In a slightly diff erent context, the delivery of suitable ultrasonic fields is necessary to achieve a uniform treatment across a therapeutic target. This thesis also reports on a study on the design of ultrasonic lenses to alter the focal region of a focussed ultrasound transducer with the aim of producing focal regions that can enable sonoporation of tumours of varying sizes. We show that the use of lenses can be an inexpensive alternative to more complex systems such as phased array transducers. Design modelling and experimental testing of lens prototypes are presented along with preliminary results with tissue mimicking polyacrylamide gel phantoms. The target environment in which the process of sonoporation will be clinically useful (i.e. in the physiological circulation) can be simpli ed as a microfluidic system. One strategy for bubble mediated therapy involves the use of a pro-drug approach, that is, when two otherwise benign ingredients are loaded onto separate microbubble populations, but can become mixed at the anatomical target site by the action of focussed ultrasound whereupon a potent drug is produced. The required mixing can be achieved by the violent coalescence of nearby cavitating bubbles, their reaction product then being released and di ffused into the interiour of nearby cells through sonoporation. A study related to this field is presented here where laser induced thermocapillary flows are shown to cause mixing of the content of a drop in a microfluidic channel in a bid to understand the mixing process at a level that may assist future microbubble engineering strategy. To summarise then, the work presented in this thesis has consolidated earlier unpublished data sets achieved by the group, providing new and exacting experimental evidence and an accurate algorithm that will facilitate post-processing of that earlier data (Chapters 2-3). Moreover, group aspirations to translate earlier in-vitro work on sonoporation towards next phase medical-phantom exposures have been boosted through the provision of a new direction involving acoustic lensing, the experimental data from which was used to completely validate existing models for our own design scenarios (Chapter 4). Finally, previous unpublished observations on microbubble coalescence undertaken by the group suggested a means to implement pro-drug delivery with direct in-situ mixing. Such suggestions were explored within microfluidic contexts using lasers to control and visualise the mixing processes that might arise in such situations (Chapter 5). All of these new insights have served to consolidate the group's previous and as yet unpublished data, opening the way for dissemination with confidence in the integrity of that data, and have also extended group capability and expertise in the areas of MHz-rate high speed framing cameras, the fabrication of acoustic lenses, and with microfluidic mixing.

Articular cartilage is a complex soft tissue covering the end of moving bones in joints which provide pressure load distribution over the joint surface and smooth lubrication with little friction for establishing movement. Articular cartilage has an intrinsically limited capacity for self-repair when injured due to the lack of nerve and blood supply. Considered that injured cartilage is left untreated, it is likely to undergo progressive cartilage degeneration without pain which may lead to posttraumatic osteoarthritis. Therefore functional and physiologic restoration of injured cartilage back to a normal condition has long been in demand, yet current available repairing methods in clinics have met with limited success. Mechanically applied loads to articular cartilage is necessary for chondrocytes, cartilage cells, since they are responsible for cartilage matrix turnover by synthesizing extracellular matrix (ECM) molecules in response to bio- chemical and mechanical changes in ECM. Ultrasound has emerged as an anabolic stimulator over the past few decades and a number of studies have proven that ultrasound therapy is beneficial for cartilage repair by synthesizing cartilage ECM components such as type II collagen and proteoglycan. Ultrasound therapy has also proven its potential for the attenuation of progressive cartilage degradation and induction of chondrogenic differentiation of mesenchymal stem cells. The use of ultrasound as an anabolic stimulator would be valuable with respect to cartilage repair since ultrasound as a form of mechanical energy can be non-invasively transferred into a human body. However, understanding the underlying mechanisms has been slow and the mechanisms have been roughly classified into thermal and non-thermal effects. Biologically detailed underlying mechanisms have not been sufficiently studied. That might be the reason why the application of ultrasound as a therapeutic tool has been limitedly available in clinics. In this study, mechanism involved biophysical effects of low intensity ultrasound has been studied for cartilage regeneration. First of all, the effect of ultrasound therapy as a mechanical stimulator on chondrogenic progenitor cell homing toward injured sites in cartilage was investigated with underlying biologic mechanisms. And the feasibility of ultrasound therapy for reactive oxygen species production mediated cartilage energy modulation was evaluated. There have been extensive preclinical studies about the effects of microbubble mediated ultrasound therapy on the targeted drugs or gene delivery into tissues of interest. Mechanical shock waves are released during ultrasound mediated microbubble destruction and the waves facilitate drug delivery into target tissues through transient blood vessel disruption. However, the clinical use of this technique has been limited through vascular system. In this study, the effects of microbubble mediated low intensity ultrasound therapy on directly delivered mechanical shock waves and controlled drug release were investigated. In conclusion, low intensity ultrasound therapy accelerates the homing of chondrogeic progenitor cells toward injured sites in cartilage via triggering mechanotransductive cell signaling pathways. This may result in speed up the return to normal cellularity and cartilage integrity by accelerating cartilage matrix repair. Low intensity ultrasound therapy was investigated as an energy modulator for chondrocytes via reactive oxygen species production in articular cartilage; however, little effects of ultrasound therapy driven cartilage energy modulation were found. The strong relationship between microbubbles mediated low intensity ultrasound therapy and the controlled release of drugs and mechanical shock waves was found. This strongly suggests that low intensity ultrasound therapy can play a role as a non-invasive controller for the release of drugs and lethal shock waves upon request.