Medical ultrasound imaging is ubiquitous in clinics due to its safety, low cost, portability, and imaging depth. The development of technologies to assist ultrasound in the diagnosis of diseases thus have a potentially broad clinical impact. More recently, photoacoustics has emerged as a complementary, high contrast modality for imaging optical absorption. Injectable dyes and nanoparticles locally amplify ultrasound and photoacoustic signal, helping to identify disease markers and track its progression. We have constructed a dual ultrasound and photoacoustic contrast agent that can be activated using an external optical trigger. In response to pulsed laser irradiation, the particle undergoes a liquid to gas phase change, or vaporization, which emits a strong acoustic wave and results in an echogenic microbubble, simultaneously enhancing contrast for both modalities. We designed and developed several iterations of particles, altering parameters to optimize biocompatibility, cost, and image contrast enhancement, and we then characterized key traits of the particles. Next, we imaged the contrast agents in phantom, ex vivo, and in vivo models to validate the image enhancement, developing image process algorithms to maximize image quality. These optically triggered contrast agents are a valuable tool for minimally invasive, highly specific, early identification of cancer. / text

Focused ultrasound therapy can be enhanced with microbubbles by thermal and cavitation effects. However, localization of treatment becomes difficult as bioeffects can occur outside of the target region. Spatial control of gas bubbles can be achieved with acoustic vaporization of perfluorocarbon droplets. This study was undertaken to determine the acoustic parameters for bubble production by droplet vaporization and how it depends on the acoustic conditions and droplet physical parameters. Droplets of varying sizes were sonicated in vitro with a focused ultrasound transducer and varying frequency and exposure. Simultaneous measurements of the vaporization and inertial cavitation thresholds were performed. The results show that droplets cannot be vaporized at low frequency without inertial cavitation occurring. However, the vaporization threshold decreased with increasing frequency, exposure and droplet size. In summary, we have demonstrated that droplet vaporization is feasible for clinically-relevant sized droplets and acoustic exposures.

Focused ultrasound therapy can be enhanced with microbubbles by thermal and cavitation effects. However, localization of treatment becomes difficult as bioeffects can occur outside of the target region. Spatial control of gas bubbles can be achieved with acoustic vaporization of perfluorocarbon droplets. This study was undertaken to determine the acoustic parameters for bubble production by droplet vaporization and how it depends on the acoustic conditions and droplet physical parameters. Droplets of varying sizes were sonicated in vitro with a focused ultrasound transducer and varying frequency and exposure. Simultaneous measurements of the vaporization and inertial cavitation thresholds were performed. The results show that droplets cannot be vaporized at low frequency without inertial cavitation occurring. However, the vaporization threshold decreased with increasing frequency, exposure and droplet size. In summary, we have demonstrated that droplet vaporization is feasible for clinically-relevant sized droplets and acoustic exposures.

Design and characterization of tissue engineered substitutes rely on robust monitoring techniques that provide information regarding viability and function when exposed to various environmental conditions. In vitro studies permit the direct monitoring of cellular and construct changes because these substitutes remain accessible. However, upon in vivo implantation, changes in cell viability and function are often detected using indirect or invasive methods that make assessing temporal changes challenging. . Thus, the development of non-invasive monitoring modalities may facilitate improved tissue substitute design and, ultimately, clinical outcome. The overall objective of this thesis was to establish a method to monitor and track cells and the cellular environment within a tissue engineered substitute in vitro and in vivo. This was accomplished via 31P NMR spectroscopy and through the incorporation of perfluorocarbon (PFC) emulsions for the monitoring of DO concentration by 19F NMR spectroscopy. The first aim of this thesis was to develop a method that tracked the state of cells and of the cellular environment within alginate constructs during perfusion studies in which the perfusing medium DO concentrations were changed over time or cells were exposed to a cytotoxic antibiotic. Due to challenges in acquiring DO concentration gradient information within beads, a second aim was to develop a mathematical model that would calculate gradients from experimentally acquired volume averaged DO concentrations; thus, significantly enhancing the robustness of tracking the alginate beads. Lastly, since the PFC emulsions used in the study may affect cell viability and function, a third aim was to characterize, experimentally and via modeling, the effect of several PFC emulsion concentrations on the encapsulated and #946;TC-tet cells.

Perfluorocarbon tracers (PFTs) are commonly proposed tracers for use in carbon capture and sequestration (CCS) leak detection and vadose zone monitoring programs. Tracers are co-injected with supercritical CO₂ and monitored in the vadose zone to identify leakage and calculate leakage rates. These calculations assume PFTs exhibit “ideal” tracer behavior (i.e. do not sorb onto or react with porous media, partition into liquid phases or undergo decay). This assumption has been brought into question by lab and field evaluations showing PFT partitioning into soil contaminants and sorbing onto clay. The objective of this study is to identify substrates in which PFTs behave conservatively and quantify non-conservative behavior. PFT breakthrough curves are compared to those of a second, conservative tracer, sulfur hexafluoride (SF₆). Breakthrough curves are generated in 1D flow-through columns packed with 5 different substrates: silica beads, quartz sand, illite, organic-rich soil, and organic-poor soil. Constant flow rate of carrier gas, N₂, is maintained. A known mass of tracer is injected at the head of the columns and the effluent analyzed at regular intervals for tracers at picogram levels by gas chromatography. PFT is expected to behave conservatively with respect to SF₆ in silica beads or quartz sand and non-conservatively in columns with clay or organics. However, results demonstrate PFT retardation with respect to SF₆ in all media (retardation factor is 1.1 in silica beads and quartz sand, 2.5 in organic-rich soil, >20 in organic-poor soil, and >100 in illite). Retardation is most likely due to sorption onto clays and soil organic matter or condensation to the liquid phase. Sorption onto clays appears to be the most significant factor. Experimental data are consistent with an analytical advection/diffusion model. These results show that PFT retardation in the vadose zone has not been adequately considered for interpretation of PFT data for CCS monitoring. These results are preliminary and do not take into account more realistic vadose zone conditions such as the presence of water, in which PFTs are insoluble. Increased moisture content will likely decrease sorption onto porous media and retardation in the vadose zone may be less than determined in these experiments. / text

Decompression sickness (DCS) results from a sudden decrease in ambient pressure leading to super-saturation of tissues with inert gas and subsequent bubble formation within both tissues and blood. Perfluorocarbons (PFC) are able to dissolve vast amounts of non-polar gases. The administration of intravenous (I.V.) PFC emulsions reduce both morbidity and mortality of DCS, but the mechanism of this protective effect has not yet been demonstrated. Juvenile Dorper cross sheep between 16 and 24 kg (n=31) were anaesthetized and instrumented for physiological monitoring, the administration of I.V. fluids and sampling of arterial and mixed venous blood. Animals were placed in a hyperbaric chamber and compressed to 6.0 atmospheres absolute for 30 minutes, then rapidly decompressed. Upon chamber exit animals were randomly assigned to receive 6cc/kg of either PFC or saline control over 5 minutes beginning immediately after chamber exit. They were also randomized to receive one of 4 breathing gases post-chamber: 100% O2, 80/20 N2/O2, 50/50 HeO2, or 80/20 HeO2. Blood samples were drawn at 5, 10, 15, 30, 60, and 90 minutes to examine whole-body oxygenation. Respiratory gases were monitored and recorded in real-time using mass spectroscopy to examine nitrogen washout. PFC administration increased arterial oxygen content (16.30±0.27 vs. 14.75±0.25 mL/dL, p<0.0001), oxygen delivery (14.83±0.28 vs. 13.44±0.25 mL/minute/kg, p=0.0004), and tissue oxygen consumption (3.37±0.14 vs. 2.76±0.13 mL/minute/kg, p=0.0018) over saline control, but did not increase mixed venous oxygen content (12.45±0.26 vs. 11.74±0.24 mL/dL, p=0.0558) or extraction ratio (0.23±0.012 vs. 0.21±0.011, p=0.1869). PFC administration lowered the plateau of the curve, increasing the amount of nitrogen washout vs. saline control (22.22±1.566 vs. 15.98±1.380 mmHg, p= 0.0074). Breathing 80/20 HeO2 increased the decay constant of the curve, increasing the rate of washout vs. breathing 100% O2 (0.03176±0.001044 vs. 0.03096±0.0009402, p=0.5777). PFC improves whole-body oxygenation after severe DCS and increases the amount of nitrogen washout. Although the effects of both PFC and 80/20 HeO2 breathing were statistically significant the magnitude of the nitrogen washout effect is quite small, and unlikely to be clinically significant. Thus it is likely that the improved oxygenation is responsible for the previously-observed therapeutic effects of PFC in treating DCS.

The purpose of the investigation was the design and development of an oxygen carrier system for oxygenation of liver cells (hepatocytes) in a bio-artificial liver support system. Acute liver failure is a devastating condition with higher than 80% mortality. Currently the only successful treatment is orthotopic liver transplantation. The high mortality rate could be reduced if a system could be developed that could bridge the patient either until recovery (due to the liver’s well-known regeneration ability) or until transplantation. Such a system requires a bioreactor with a high density of cultured cells. Sufficient oxygen delivery to the cells is critical to ensure efficient cell function. The CSIR and University of Pretoria (UP) have designed and developed a novel bio-artificial liver support system (BALSS) that utilizes perfluorooctyl bromide (PFOB) as artificial oxygen carrier. As the PFOB is not miscible with water, it needs to be emulsified. To enable the use of the PFOB emulsion in the UP-CSIR BALSS, a study was carried out to investigate relevant aspects relating to the PFOB emulsion, i.e. the formulation, manufacturing procedure, stability, rheology and mass transfer characteristics. The study results are reported in this dissertation, including a proposed mass transfer model for describing oxygen mass transfer to and from the PFOB emulsions. Emulsion stability can be improved through control of the droplet size and size distribution, limiting Ostwald ripening, and control of zeta potential of the dispersed phase droplets. PFOB emulsions with dispersed phase (PFOB) volume fractions between 0.4 and 0.5 and Sauter mean droplet diameter between 100 and 200 nm were found to be optimal for oxygen mass transfer in cell culture systems. The PFOB emulsion in the UP-CSIR BALSS can be concentrated and recirculated using ultrafiltration. Quantitative recovery of PFOB from its emulsions can be carried out using distillation with orthophosphoric acid. Experimental overall mass transfer coefficients for membrane oxygenators obtained without PFOB compared well with literature reported values of 2.5x10-5 m/s by Goerke et al. (2002) and 1 – 3x10-5 m/s by Schneider et al. (1995) for similar systems. The addition of 0.2 v/v PFOB leads to an increase in the membrane oxygenator mass transfer coefficient by a factor of about 30, and an increase in oxygen carrying capacity by a factor of about 4.5. It was also shown that suitable PFOB emulsions can have a significant impact on the growth and function of hepatocytes in a BALSS. / Thesis (PhD (Chemical Engineering))--University of Pretoria, 2005. / Chemical Engineering / unrestricted

Au cours de la ventilation liquide totale (VLT), les cellules pulmonaires sont exposées à des perfluorocarbones (PFC) dont les propriétés physiques diffèrent fortement du milieu standard de culture cellulaire (DMEM) et encore plus des propriétés de l'air. Dans cette thèse nous étudions les effets d’une exposition au PFC sur la réponse des cellules épithéliales pulmonaires, en effectuant une étude approfondie des propriétés structurales, mécaniques et fonctionnelles. La réponse des cellules A549 (alvéolaire), HBE (bronchique) et AM (Macrophage alvéolaire) exposées au PFC est étudiée par comparaison au DMEM. Les variations de la structure de F-actine, de la densité d'adhésion focale et de la distribution du glycocalyx sont évaluées par fluorescence. Les changements de propriétés mécaniques et de paramètres d’adhésion sont mesurés par la Magnétocymétrie (MTC) étendue à l’analyse multiéchelle. La mécanique cellulaire est caractérisée par deux modèles microrhéologiques reflétant deux types de comportement possibles du cytosquelette (CSK). L'adhésion à la matrice cellulaire est analysée par un modèle stochastique de dé-adhésion, décrivant la composante non-réversible de la réponse cellulaire. Les rôles fondamentaux de la structure de F-actine et de la couche de glycocalyx sont respectivement évalués par dépolymérisation de F-actine et en dégradant le glycocalyx. Les résultats montrent que l'exposition au PFC induit un remodelage de la structure de F-actine, un affaiblissement du CSK et une diminution de l'adhésion. Ces résultats démontrent que le PFC déclenche une réponse particulière des cellules épithéliales caractérisée par une diminution de la tension intracellulaire, l'affaiblissement de l'adhésion et la redistribution du glycocalyx. L’origine de cette adaptation cellulaire est physique et très probablement reliée à l’augmentation de l'énergie interfaciale associée à la basse tension de surface d’un PFC chimiquement apolaire. La faible tension de surface du PFC est également responsable d'une augmentation de la compliance pulmonaire pendant VLT et a des impacts profonds dans les paramètres respiratoires, parallèlement à la modification de la réponse cellulaire. / During Total Liquid Ventilation (TLV), lung cells are exposed to perfluorocarbon (PFC) whose physical properties highly differ from aqueous medium (DMEM) standardly used for cell culture and farther air properties. In this thesis, we study the effects of PFC exposure on the response of pulmonary epithelial cell by performing a thorough assessment of their structural, mechanical and functional properties. The response of A549 cells (alveolar), HBE (bronchial), and AM (alveolar macrophages) exposed to PFC is studied by comparison to DMEM. Changes in F-actin structure, focal adhesion size and density and glycocalyx expression are evaluated by fluorescence. Changes in cell mechanics and adhesion parameters are measured by a multiscale Magnetic Twisting Cytometry (MTC) method. Cell mechanics is analyzed by two microrheological models reflecting two possible cytoskeleton features. Cell-matrix adhesion is analyzed by a stochastic multibond de-adhesion model describing the non-reversible component of the cell response by MTC. The key roles of F-actin structure and glycocalyx layer are established by respectively depolymerising F-actin and degrading glycocalyx. Results show that PFC exposure induces F-actin remodelling, cytoskeleton softening and adhesion weakening. They demonstrate that PFC triggers an epithelial cell response which is characterized by decay in intracellular tension, adhesion weakening and glycocalyx redistribution. The origin of this cellular adaptations is physical and most likely related to the increase in interfacial energy, associated to the low surface tension of the non polar perflurorocarbon, The low surface tension of PFC is also responsible for an increase in lung compliance during TLV and has deep impacts during ventilation parallel to the modification of cell response.

Personalized medicine can be an approach to address the unsatisfactory treatment outcomes in inflammatory conditions such as cancer, arthritis, and cardiovascular diseases. A common feature of chronic diseases is the infiltration of pro-inflammatory macrophages at the disease loci. Infiltrating macrophages have been previously utilized for disease diagnosis. These features suggest that macrophages can be broadly applicable targets for simultaneous therapy and diagnosis. Cyclooxygenase-2 (COX-2), an enzyme involved in the biosynthesis of a lipid inflammatory mediator, prostaglandin E2 (PGE2), is over expressed in macrophages infiltrating the pathological site. Inhibition of PGE2 leads to reduced inflammation, pain and macrophage infiltration. To utilize macrophages for the purpose of simultaneous therapy and diagnosis, we proposed to integrate therapeutic and imaging capabilities on a single nanomedicine platform, referred as theranostics. A stable 19F MRI visible nanoemulsion platform was developed, incorporating celecoxib for COX-2 inhibition and near-infrared fluorescent dye(s) for fluorescence imaging. We hypothesized that inhibition of COX-2 in macrophages using a theranostic nanoemulsion will reduce the inflammation (and pain), and that this response can be visualized by monitoring changes in macrophage infiltration. In vitro characterization demonstrated that the theranostic displays excellent stability with no toxicity, and significant uptake in macrophages. Furthermore, it delivers celecoxib to macrophages and reduces PGE2 production from these cells. In vivo studies in a murine paw inflammation model showed nanoemulsion presence at the inflamed site, specifically in COX-2 expressing macrophages compared to neutrophils. Supporting our hypothesis, celecoxib delivered through a nanoemulsion demonstrated time-dependent reduction in fluorescence from the inflamed paw, indicative of reduced macrophage infiltration. In a neuropathic pain model, celecoxib delivered to macrophages led to reduced pain concomitant with reduced macrophage infiltration at the inflamed site compared to free drug control (cross reference: Kiran Vasudeva, Dissertation, 2015). In conclusion, inhibition of COX-2 in macrophages using theranostic nanoemulsions proves to be an effective and generalized strategy facilitating simultaneous therapy and diagnosis, which can be applied to many chronic diseases. The diagnostic information during therapy can be used to tailor the treatment and reduce patient variability leading to personalized medicine. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmaceutics / PhD; / Dissertation;

Implantation of tissue engineered pancreatic constructs can provide for a physiologic regulation of blood glucose levels. A major concern in designing such constructs is ensuring sufficient oxygenation of the cells, as oxygen is usually the limiting nutrient affecting cell viability and function. Furthermore, in vivo factors influencing construct oxygenation often lead to implant failure, and are detected primarily on end physiologic effects. The ability of perfluorocarbons (PFCs) to dissolve large amounts of oxygen and their high fluorine content makes these compounds a potentially valuable oxygen delivery tool and good 19F Nuclear Magnetic Resonance (NMR) markers for dissolved oxygen concentration (DO). Experimental studies and simulations showed that although the addition of 10 vol% PFC increased construct oxygenation, this improvement was minimal and had limited benefits on the growth and function of encapsulated bTC-tet cells under normoxic and hypoxic conditions. A dual PFC method that utilizes 19F NMR spectroscopy was developed to noninvasively monitor DO within a tissue construct and in its surroundings. In vitro studies using an NMR-compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active bTC-tet cells, relative to the DO in the culture medium, under perfusion and static conditions. In vivo, the method was capable of acquiring real-time DO measurements in murine models. Measured DO can be correlated with the physiological state of the implant examined post-explantation and was compatible with the therapeutic function of the implant.