Engineering Lipid-stabilized Microbubbles for Magnetic Resonance Imaging guided Focused Ultrasound Surgery

Lipid-stabilized microbubbles are gas-filled microspheres encapsulated with a phospholipid monolayer shell. Because of the high echogenicity provided by its highly compressible gas core, these microbubbles have been adapted as ultrasound contrast agents for a variety of applications such as contrast-enhanced ultrasonography (CEUS), targeted drug delivery and metabolic gas transport. Recently, these lipid-stabilized microbubbles have demonstrated increased potential as theranostic (therapy + diagnostics) agents for non-invasive surgery with focused ultrasound (FUS). For instance, their implementation has reduced the acoustic intensity threshold needed to open the blood-brain-barrier (BBB) with FUS, which potentially allows for the localized delivery of drugs to treat neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. However, the effectiveness of microbubbles for this application is dependent on successful microbubble engineering. One necessary improvement is the development and utilization of monodisperse microbubbles of varying size classes. Another design improvement is the development of a microbubble construct whose fragmentation state during or after FUS surgery can be tracked by magnetic resonance imaging (MRI). Thus, in this thesis, we describe a method to generate and select lipid-coated gas-filled microbubbles of specific size fractions based on their migration in a centrifugal field. We also detail the design and characterization of size-selected lipid-coated microbubbles with shells containing the magnetic resonance (MR) contrast media Gadolinium (Gd(III)), for utility in both MR and ultrasound imaging. Initial characterization of the lipid headgroup labeled Gd(III)-microbubbles by MRI revealed that the Gd(III) relaxivity increased after microbubble fragmentation into non-gas-containing lipid vesicles. This behavior was explained to stem from an increase in interaction between water protons and the Gd(III)-bound lipid fragments due to an increase in lipid headgroup area after microbubble fragmentation. To explore this hypothesis, an alternative construct consisting of Gd(III) preferentially bound to the protective poly(ethylene glycol) (PEG) brush of the lipid shell architecture was also designed and compared to the lipid headgroup-labeled Gd(III)-microbubbles. Nuclear magnetic resonance (NMR) analysis revealed that, in contrast to the headgroup labeled Gd(III)-microbubbles, the relaxivity of the PEG-labeled Gd(III)-microbubbles decreased after microbubble fragmentation. NMR analysis also revealed an independent concentration-dependent enhancement of the transverse MR signal by virtue of the microbubble gas core. The results of this study illustrated the roles that Gd(III) placement on the lipid shell and the presence of the gas core may play on the MR signal when monitoring Gd(III)-microbubble cavitation during non-invasive surgery with FUS

Evaluation of Peritoneal Microbubble Oxygenation Therapy in a Rabbit Model of Hypoxemia

Alternative extrapulmonary oxygenation technologies are needed to treat patients suffering from severe hypoxemia refractory to mechanical ventilation. We previously demonstrated that peritoneal microbubble oxygenation (PMO), in which phospholipid-coated oxygen microbubbles (OMBs) are delivered into the peritoneal cavity, can successfully oxygenate rats suffering from a right pneumothorax. This study addressed the need to scale up the procedure to a larger animal with a splanchnic cardiac output similar to humans. Our results show that PMO therapy can double the survival time of rabbits experiencing complete tracheal occlusion from6.6 ± 0.6 min for the saline controls to 12.2 ± 3.0 min for the bolus PMO-treated cohort. Additionally, we designed and tested a new peritoneal delivery system to circulate OMBs through the peritoneal cavity. Circulation achieved a similar survival benefit to bolus delivery under these conditions. Overall, these results support the feasibility of the PMO technology to provide extrapulmonary ventilation for rescue of severely hypoxic patients

Reducing Tumour Hypoxia via Oral Administration of Oxygen Nanobubbles

Hypoxia has been shown to be a key factor inhibiting the successful treatment of solid tumours. Existing strategies for reducing hypoxia, however, have shown limited efficacy and/or adverse side effects. The aim of this study was to investigate the potential for reducing tumour hypoxia using an orally delivered suspension of surfactant-stabilised oxygen nanobubbles. Experiments were carried out in a mouse xenograft tumour model for human pancreatic cancer (BxPc-3 cells in male SCID mice). A single dose of 100 μL of oxygen saturated water, oxygen nanobubbles or argon nanobubbles was administered via gavage. Animals were sacrificed 30 minutes post-treatment (3 per group) and expression of hypoxia-inducible-factor-1α (HIF1α) protein measured by real time quantitative polymerase chain reaction and Western blot analysis of the excised tumour tissue. Neither the oxygen saturated water nor argon nanobubbles produced a statistically significant change in HIF1α expression at the transcriptional level. In contrast, a reduction of 75% and 25% in the transcriptional and translational expression of HIF1α respectively (p<0.001) was found for the animals receiving the oxygen nanobubbles. This magnitude of reduction has been shown in previous studies to be commensurate with an improvement in outcome with both radiation and drug-based treatments. In addition, there was a significant reduction in the expression of vascular endothelial growth factor (VEGF) in this group and corresponding increase in the expression of arrest-defective protein 1 homolog A (ARD1A)

Noninvasive, Transient and Selective Blood-Brain Barrier Opening in Non-Human Primates In Vivo

The blood-brain barrier (BBB) is a specialized vascular system that impedes entry of all large and the vast majority of small molecules including the most potent central nervous system (CNS) disease therapeutic agents from entering from the lumen into the brain parenchyma. Microbubble-enhanced, focused ultrasound (ME-FUS) has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. For the first time, the feasibility of transcranial ME-FUS BBB opening in non-human primates is demonstrated with subsequent BBB recovery. Sonications were combined with two different types of microbubbles (customized 4–5 µm and Definity®). 3T MRI was used to confirm the BBB disruption and to assess brain damage

In Vitro Acoustic Characterization of Three Phospholipid Ultrasound Contrast Agents from 12 to 43 MHz

AbstractThe acoustic properties of two clinical (Definity, Lantheus Medical Imaging, North Billerica, MA, USA; SonoVue, Bracco S.P.A., Milan, Italy) and one pre-clinical (MicroMarker, untargeted, Bracco, Geneva, Switzerland; VisualSonics, Toronto, ON, Canada) ultrasound contrast agent were characterized using a broadband substitution technique over the ultrasound frequency range 12–43 MHz at 20 ± 1°C. At the same number concentration, the acoustic attenuation and contrast-to-tissue ratio of the three native ultrasound contrast agents are comparable at frequencies below 30 MHz, though their size distributions and encapsulated gases and shells differ. At frequencies above 30 MHz, native MicroMarker has higher attenuation values and contrast-to-tissue ratios than native Definity and SonoVue. Decantation was found to be an effective method to alter the size distribution and concentration of native clinical microbubble populations, enabling further contrast enhancement for specific pre-clinical applications

Focused ultrasound-mediated intranasal brain drug delivery technique (FUSIN)

The blood-brain barrier (BBB) is the major obstacle for brain drug delivery and limits the treatment options for central nervous system diseases. To circumvent the BBB, we introduce focused ultrasound-mediated intranasal brain drug delivery (FUSIN). FUSIN utilizes the nasal route for direct nose-to-brain drug administration, bypassing the BBB and minimizing systemic exposure to the major organs, such as heart, lung, liver, and kidney [1]. It also uses transcranial ultrasound energy focused at a targeted brain region to induce microbubble cavitation, enhancing the transport of intranasally administered agents at the FUS-targeted brain location. FUSIN is unique because it can achieve noninvasive and localized brain drug delivery with minimized systemic toxicity to other major organs. The goal of this paper is to provide a detailed protocol for FUSIN delivery to the mouse brain. • FUSIN delivery utilizes the nose-to-brain pathway for brain drug delivery. • FUSIN utilizes FUS combined with microbubble to significantly enhance the delivery efficiency of intranasally administered drugs to the FUS targeted brain regions. • FUSIN achieves efficient brain delivery with minimized systemic exposure in the major organs