Thesis advisor: Cyril P. Opeil / The physiology of the vasculature in the central nervous system (CNS) which includes the blood-brain-barrier (BBB) and other factors, prevents the transport of most anticancer agents to the brain and restricts delivery to infiltrating brain tumors. The heterogeneous vascular permeability in tumor vessels (blood-tumor barrier; BTB), along with several other factors, creates additional hurdles for drug treatment of brain tumors. Different methods have been used to bypass the BBB/BTB, but they have their own limitations such as being invasive, non-targeted or requiring the formulation of new drugs. Magnetic Resonance Imaging guided Focused Ultrasound (MRIgFUS), when combined with circulating microbubbles, is an emerging noninvasive method to temporarily permeabilize the BBB and BTB. The purpose of this thesis was to use this alternative approach to deliver chemotherapeutic agents through the BBB/BTB for brain tumor treatment in a rodent model to overcome the hinderances encountered in prior approaches tested for drug delivery in the CNS. The results presented in thesis demonstrate that MRIgFUS can be used to achieve consistent and reproducible BBB/BTB disruption in rats. It enabled us to achieve clinically-relevant concentrations of doxorubicin (~ 4.8±0.5 µg/g) delivered to the brain with the sonication parameters (0.69 MHz; 0.55 MPa; 10 ms bursts; 1 Hz PRF; 60 s duration), microbubble concentration (Definity, 10 µl/kg), and liposomoal doxorubicin (Lipo-DOX) dose (5.67 mg/kg) used. The resulting doxorubicin concentration was reduced by 32% when the agent was injected 10 minute after the last sonication. Three weekly sessions of FUS and Lipo-DOX appeared to be safe in the rat brain, despite some minor tissue damage. Importantly, the severe neurotoxicity seen in earlier works using other approaches does not appear to occur with delivery via FUS-BBB disruption. The resuls from three weekly treatments of FUS and Lipo-DOX in a rat glioma model are highly promising since they demonstrated that the method significantly inhibits tumor growth and improves survival. Animals that received three weekly sessions of FUS + Lipo-DOX (N = 8) had a median survival time that was increased significantly (P<0.001) compared to animals who received Lipo-DOX only (N = 6), FUS only (N = 8), or no treatment (N = 7). Median survival for animals that received FUS + Lipo-DOX was increased by 100% relative to untreated controls, whereas animals who received Lipo-DOX alone had only a 16% improvement. Animals who received only FUS showed no improvement. No tumor cells were found in histology in 4/8 animals in the FUS + Lipo-DOX group, and only a few tumor cells were detected in two animals. Tumor doxorubicin concentrations increased monotonically (823±600, 1817±732 and 2432±448 ng/g) in the control tumors at 9, 14 and 17 days respectively after administration of Lipo-DOX. With FUS-induced BTB disruption, the doxorubicin concentrations were enhanced significantly (P<0.05, P<0.01, and P<0.0001 at days 9, 14, and 17, respectively) and were greater than the control tumors by a factor of two or more (2222±784, 3687±796 and 5658±821 ng/g) regardless of the stage of tumor growth. The transfer coefficient Ktrans was significantly (p<0.05) enhanced compared to control tumors only at day 9 but not at day 14 or 17. These results suggest that FUS-induced enhancements in tumor drug delivery for Lipo-DOX are relatively consistent over time, at least in this tumor model. These results are encouraging for the use of large drug carriers, as they suggest that even large/late-stage tumors can benefit from FUS-induced drug enhancement. Corresponding enhancements in Ktrans were found variable in large/late-stage tumors and not significantly different than controls, perhaps reflecting the size mismatch between the liposomal drug (~100 nm) and Gd-DTPA (molecular weight: 938 Da). Overall, this thesis research provides pre-clinical data toward the development of MRIgFUS as a noninvasive method for the delivery of agents such as Lipo-DOX across the BBB/BTB to treat patients with diseases of the central nervous system. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Physics.

This study presents a systematic investigation of two types of soft biomaterials: phospholipid-based microbubbles (MBs) and agarose hydrogels, using atomic force microscopy (AFM) force-distance curves. Microbubbles are used widely in several applications, especially in medical applications, where they are used as ultrasound contrast agents (UCAs) and as vehicles for transporting the drugs and genes to their targets, which is commonly known as drug/gene delivery. Although plenty of attention has been paid to these materials by medical researchers there is a shortage of engineering research on the properties of these materials. The present study tries to address this gap by studying these materials from the engineering perspective; therefore, the aim of this study is to investigate the mechanical properties of MBs and hydrogels. In this research, phospholipid-based microbubbles (MBs), commercially called SonoVue® microbubbles and used as UCAs, were investigated to measure their mechanical properties using an AFM mode of operation called force-distance curves (or force spectroscopy mode); this mode allows for direct mechanical tests to acquire the force-deformation (F-Δ) behaviour of the MBs. The compression tool was a flat (tipless) cantilever moved at constant speed, whereas the variable was MB size. The MBs behaviour was assessed by calculating several mechanical properties, which were the stiffness, Young's modulus (three different models were applied), hysteresis, plasticity, adhesion forces, nonlinearity and instability. The stiffness and the Young's modulus values were measured to be in the same range as found in similar studies. A phenomenon was observed that the local stiffness of the MB increases after each unstable step provided that the MB stays within the linear elastic region. The Young's modulus was calculated applying three models, two for estimating the elastic modulus of the shell and the third for modulus of elasticity of the whole MB. The stretching component of the membrane theory was found to provide the best prediction of the Young's modulus value. To investigate the effect of the tip geometry on the mechanical properties of the MBs, the MBs were studied with different cantilever/tips, including a conical-tipped cantilever. The study concluded that there is no impact of the contact geometry on the mechanical properties of the MBs if the applied forces and the spring constant of the cantilever are the same. The same phenomenon, increasing the local stiffness of the MB after each unstable step, was found however with a higher rate. Hydrogels were also studied in this research using AFM and adopting a nanoindentation technique. The indenter was a conical tip moving toward the sample surface with constant speed and applying similar forces on all samples, where the variable was the gel concentration. In addition to the previous mechanical properties, other properties were investigated, such as hardness, universal hardness and pressure. An effect of the gel concentration on the mechanical properties of the gels was observed. There is a difference in the results compared to those reported in the literature review, where some of the results are in the same range as those found here, while others were either higher or lower, due to the influence of factors such as the indenter geometry, the applied force and the load rate; moreover, it was found that the viscoelastic behaviour of the gels played a significant role.

Revascularization of Chronic Total Occlusions (CTO) by percutaneous coronary interventions is limited by low success rates, primarily due to difficulty in guidewire crossing. There are a number of contributing factors that make guidewire crossing challenging. Two of the most significant impediments are: a) inability to adequately visualize the CTO to appropriately plan a pathway to the distal lumen, and b) difficulty in physically crossing the rigid endcap at the proximal end of CTO without using stiff wires. Moreover, there is a significant knowledge gap in the composition of CTOs, and the consequent impact of that composition on crossability. This thesis presents tools and techniques to help mitigate the current shortcomings, while shedding new light on CTO composition and maturation. The tools and techniques presented herein are based upon ultrasound approaches with the intent of eventually developing these strategies into catheter based solutions. Recent studies have suggested that the presence of microvessels in CTO may provide a preferred pathway for guidewire crossing. However, due to limited resolution and a lack of soft tissue contrast in angiography, microvessels within CTO cannot generally be detected by in-vivo angiographic techniques, and when they are visualized, it is unknown whether or not they are intraluminal. In this thesis, high frequency ultrasound with Power Doppler overlays is shown to be capable of detecting and tracking transluminal recanalization channels using an in vivo porcine model of CTO. It is also shown that ultrasound is a more sensitive technique to detect and map these channels than MRI. Furthermore, features of microvasculature in CTOs that had not previously been seen are presented. A technique was then developed to facilitate guidewire crossing through the proximal endcap, also known as the proximal fibrous cap (PFC). In order to assess the ease with which a probe is able to iv perforate the PFC, a system was designed and to measure the force required for PFC puncture. This system was validated by examining the required puncture forces for CTOs of different ages. It was shown that CTOs less than 6 weeks in age are significantly easier to puncture than those greater than 12 weeks. This coincides with differences in composition, with the presence of softer materials at the earlier time point, such as thrombus and proteoglycans compared to stiffer fibrotic materials which predominate at late timepoints. After development and validation of a reliable technique to measure ease of PFC puncture, the efficacy of therapies designed to modify PFC compliance could be assessed. The use of ultrasound mediated microbubble (UMM) disruption to act as an adjuvant to accelerate collagenase therapy in CTO was examined. A significant reduction in puncture force and an increase in the amount of collagen degraded was achieved using a combined UMM + collagenase treatment compared with collagenase therapy alone and UMM treatment alone.

Revascularization of Chronic Total Occlusions (CTO) by percutaneous coronary interventions is limited by low success rates, primarily due to difficulty in guidewire crossing. There are a number of contributing factors that make guidewire crossing challenging. Two of the most significant impediments are: a) inability to adequately visualize the CTO to appropriately plan a pathway to the distal lumen, and b) difficulty in physically crossing the rigid endcap at the proximal end of CTO without using stiff wires. Moreover, there is a significant knowledge gap in the composition of CTOs, and the consequent impact of that composition on crossability. This thesis presents tools and techniques to help mitigate the current shortcomings, while shedding new light on CTO composition and maturation. The tools and techniques presented herein are based upon ultrasound approaches with the intent of eventually developing these strategies into catheter based solutions. Recent studies have suggested that the presence of microvessels in CTO may provide a preferred pathway for guidewire crossing. However, due to limited resolution and a lack of soft tissue contrast in angiography, microvessels within CTO cannot generally be detected by in-vivo angiographic techniques, and when they are visualized, it is unknown whether or not they are intraluminal. In this thesis, high frequency ultrasound with Power Doppler overlays is shown to be capable of detecting and tracking transluminal recanalization channels using an in vivo porcine model of CTO. It is also shown that ultrasound is a more sensitive technique to detect and map these channels than MRI. Furthermore, features of microvasculature in CTOs that had not previously been seen are presented. A technique was then developed to facilitate guidewire crossing through the proximal endcap, also known as the proximal fibrous cap (PFC). In order to assess the ease with which a probe is able to iv perforate the PFC, a system was designed and to measure the force required for PFC puncture. This system was validated by examining the required puncture forces for CTOs of different ages. It was shown that CTOs less than 6 weeks in age are significantly easier to puncture than those greater than 12 weeks. This coincides with differences in composition, with the presence of softer materials at the earlier time point, such as thrombus and proteoglycans compared to stiffer fibrotic materials which predominate at late timepoints. After development and validation of a reliable technique to measure ease of PFC puncture, the efficacy of therapies designed to modify PFC compliance could be assessed. The use of ultrasound mediated microbubble (UMM) disruption to act as an adjuvant to accelerate collagenase therapy in CTO was examined. A significant reduction in puncture force and an increase in the amount of collagen degraded was achieved using a combined UMM + collagenase treatment compared with collagenase therapy alone and UMM treatment alone.

Despite chronic dialysis treatment, patients with end stage renal disease undergoing maintenance haemodialysis (HD) remain at a substantially increased risk of morbidity. Previous reports using Doppler ultrasound (DU) during HD have revealed microembolic signals (ME) in the venous circulation. In vitro studies confirm the emergence of microbubbles of air that may pass the security system of the HD circuit without triggering the alarm. The aim of this thesis was to elucidate the presence of ME during HD and examine methods that might reduce exposure to ME in vivo. The first study utilized DU to verify the presence of ME in 40 patients during standard HD. Investigation within 30 minutes after the start of HD and just before the end of session revealed the presence of ME in the venous blood line during both phases. The air trap did not alert for the presence of ME. This indicated that ME may pass into the patient during the entire HD run. Study 2 analyzed the presence of ME prior to start and during HD when measured at the AV-access and also carotid artery. A total of 54 patients were examined using DU as the investigative technique. ME increased significantly after start of HD in the AV-access, but also at the carotid artery site. These data indicated that ME can enter the body and even pass the lung barrier. The question arose if microbubbles of air are resorbed or may cause ischemic lesions in organs such as the brain. Study 3 examined whether the amount of ME detected in the AV-access would change by using either a high or a low blood level in the venous air trap/chamber. This was a prospective, randomized and double-blind study of 20 HD patients who were their own controls. After 30 min of standard HD, measurement of ME with DU was performed for two minutes. The chamber setting was changed and after another 30 minutes a new recording was carried out for two minutes. Data showed that setting a high blood level significantly reduced the extent of ME that entered the patient. The results also indicated that ME consisted mainly of microbubbles. In study 4, twenty patients were randomized in a cross-over setting of HD. Three options were used: a wet-stored dialyzer with high blood level (WH) and a dry-stored dialyzer using either a high (DH) or a low (DL) blood level in the venous chamber. The exposure of ME, detected by DU, was least when using mode WF, more with mode DH, and most with mode DL. There was a correlation between higher blood flow and more extensive exposure to ME. Study 5 was an autopsy study of a chronic HD patient with the aim of searching for microbubbles deposited in organs. Microbubbles of gas were verified in the vessels of the lungs, brain and heart. By using a fluorescent stain of anti-fibrinogen it was verified that the microbubbles were covered by clots that had to be preformed before death occurred. This indicated that air microbubbles are not completely absorbed and could result in embolic deposition in the organs of HD patients. In conclusion, these in vivo studies showed that ME pass the air trap without inducing an alarm and enter the venous blood line of the patient. The data confirmed the presence of ME in the AV-access and also in the carotid artery. Autopsy data of a deceased HD patient demonstrated the presence of microbubbles in the capillaries of the lungs, but also in the systemic circulation such as in the brain and the heart. A high blood level in the venous chamber and wet-stored dialyzer can reduce, but not eliminate the exposure to microbubbles for patients undergoing HD.

An experimental study on drag reduction by injection of microbubbles was performed in the upper wall of a rectangular channel at Re = 5128. Particle Image Velocimetry measurement technique (PIV) was used to obtain instantaneous velocity fields in the x-y plane. Microbubbles, with an average diameter of 30??m, were produced by electrolysis using platinum wires with a diameter of 76 ??m. They were injected in the buffer layer producing several different values of local void fraction. A maximum drag reduction of 38.45% was attained with a local void fraction of 4.8 %. The pressure drop in the test station was measured by a reluctance pressure transducer. Several parameters such as velocity profile, turbulent intensities, skewness, flatness, joint probability density function (JPDF), enstrophy, one and two-dimensional energy spectra were evaluated. The results indicate that microbubbles reduced the intermittency of the streamwise fluctuating component in the region near the wall. At the same time they destroy or reduce the vortical structures regions (high shear zones) close to the wall. They also redistribute the energy among different eddy sizes. An energy shift from larger wavenumbers to lower wavenumbers is observed in the near wall region (buffer layer). However, outside this region, the opposite trend takes place. The JPDF results indicate that there is a decrease in the correlation between the streamwise and the normal fluctuating velocities, resulting in a reduction of the Reynolds stresses. The results of this study indicate that pursuing drag reduction by injection of microbubbles in the buffer layer could result in great saving of energy and money. The high wavenumber region of the one dimensional wavenumber spectra was evaluated from PIV spatial information, where the maximum wavenumber depends on the streamwise length (for streamwise wavenumber) of the recorded image and the minimum wavenumber depends on the distance between vectors. On the other hand, the low wavenumber region was calculated from the PIV temporal information by assuming Taylor??s frozen hypothesis. This new approach allows obtaining the energy distribution of a wider wavenumber region.

Encapsulated microbubbles, whose diameters are on the order of microns, are widely used to provide acoustic contrast in biomedical applications. But well below the resonance frequencies of these microbubbles, any acoustic contrast is due solely to their relatively high compressibility compared to the surrounding medium. To estimate how well microbubbles may function as acoustic contrast agents in applications such as borehole logging or underground flow mapping, it must be determined how they behave both at atmospheric and down-well conditions, and how their presence affects the bulk acoustic properties of the surrounding medium, most crucially its specific acoustic impedance. Resonance tube experiments were performed on several varieties of acoustic contrast agents to determine their compressibility as a function of pressure and temperature, and the results are used to estimate the effect on sound propagation when they are introduced into rock formations. / text

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Targeted imaging with microbubbles may resolve the molecular expression within the abnormal blood vessels of tumours. Optimal imaging requires understanding the interaction between targeted microbubbles and high-frequency ultrasound. Therefore, the subharmonic signal, and backscattering cross-section of individual bound microbubbles were examined with coaligned 30 MHz pulses and optical images. The peak subharmonic signal was generated for 1.6 µm diameter microbubbles for 20% and 11% bandwidth pulses and 1.8 µm for 45% bandwidth pulses at 200 kPa, consistent with estimations of the resonant size of microbubble's at 15 MHz. In order to measure the scattering cross-section, a new method was proposed to measure the receive transfer function of a transducer. Measurements of the backscattering cross-section scaled with the square of the radius, with signi cant size-independent variability. The results of this thesis will help optimise the parameters for targeted imaging, as well as further our understanding of the behaviour of microbubbles.

Targeted imaging with microbubbles may resolve the molecular expression within the abnormal blood vessels of tumours. Optimal imaging requires understanding the interaction between targeted microbubbles and high-frequency ultrasound. Therefore, the subharmonic signal, and backscattering cross-section of individual bound microbubbles were examined with coaligned 30 MHz pulses and optical images. The peak subharmonic signal was generated for 1.6 µm diameter microbubbles for 20% and 11% bandwidth pulses and 1.8 µm for 45% bandwidth pulses at 200 kPa, consistent with estimations of the resonant size of microbubble's at 15 MHz. In order to measure the scattering cross-section, a new method was proposed to measure the receive transfer function of a transducer. Measurements of the backscattering cross-section scaled with the square of the radius, with signi cant size-independent variability. The results of this thesis will help optimise the parameters for targeted imaging, as well as further our understanding of the behaviour of microbubbles.