Engineering responsive liposome systems for biomedical applications

The design of materials able to undergo changes in response to an applied stimulus (e.g. temperature, pH or magnetic fields) is relevant for biomedical applications. In the context of hydrogels, the design of triggers for hydrogelation has enabled precise control over hydrogelation kinetics and mechanical properties. One trigger that has yet to be explored for hydrogelation is ultrasound; a widely-used biomedical platform that is non-invasive, with tuneable tissue penetration depth and high spatiotemporal control. The use of ultrasound as a remote trigger for enzymatic activity and hydrogelation was explored in this thesis. In particular, the ability of liposomes and microbubble-liposome conjugates to release encapsulated payloads upon ultrasound exposure was leveraged. The designed field-responsive system required that the amount of encapsulated calcium in liposomes was maximised. Hence, cryo-TEM and small-angle neutron scattering were used to investigate the effect of the formulation method and the lipid composition on vesicle lamellarity, which determines the volume of the internal liposomal aqueous compartment. In another study, X-ray and neutron scattering corroborated with all-atom molecular dynamics simulations were used to elucidate the effect of sodium and calcium ions on ethanol-induced lipid membrane interdigitation. The results of this study, which spanned a wide range of length scales, furthered the understanding of ethanol-induced interdigitation of bulk and vesicular lipid formulations, with important implications for the production of interdigitation-fusion vesicles. Calcium-loaded liposomes produced via the interdigitation fusion vesicle method that were able to release their payload upon ultrasound exposure were utilised to trigger the catalytic activity of a calcium-dependent tissue transglutaminase. The ultrasound-activated transglutaminase could then catalyse intermolecular covalent crosslinking between the lysine and glutamine sidechain residues of soluble fibrinogen molecules, yielding fibrinogen hydrogels. Precise control over these processes could be achieved, with the calcium release, catalysis rate and hydrogelation rate all shown to be dependent upon the ultrasound exposure time. Calcium-loaded liposomes were also conjugated to the surface of gaseous microbubbles that are commonly used for in vivo drug delivery. These microbubble-liposome conjugates exhibited enhanced response to the applied acoustic field and could also be used for ultrasound-triggered hydrogelation. Taken together, these results represent an entirely new class of stimuli for enzyme activity and hydrogelation and open up a wide range of opportunities for ultrasound-triggered molecular biology, synthetic biology and material science.Open Acces

Vocal fold vibratory and acoustic features in fatigued Karaoke singers

Session 3aMU - Musical Acoustics and Speech Communication: Singing Voice in Asian CulturesKaraoke is a popular singing entertainment particularly in Asia and is gaining more popularity in the rest of world. In Karaoke, an amateur singer sings with the background music and video (usually guided by the lyric captions on the video screen) played by Karaoke machine, using a microphone and an amplification system. As the Karaoke singers usually have no formal training, they may be more vulnerable to vocal fatigue as they may overuse and/or misuse their voices in the intensive and extensive singing activities. It is unclear whether vocal fatigue is accompanied by any vibration pattern or physiological changes of vocal folds. In this study, 20 participants aged from 18 to 23 years with normal voice were recruited to participate in an prolonged singing task, which induced vocal fatigue. High speed laryngscopic imaging and acoustic signals were recorded before and after the singing task. Images of /i/ phonation were quantitatively analyzed using the High Speed Video Processing (HSVP) program (Yiu, et al. 2010). It was found that the glottis became relatively narrower following fatigue, while the acoustic signals were not sensitive to measure change following fatigue. © 2012 Acoustical Society of Americapublished_or_final_versio

Advances in Bioengineering

The technological approach and the high level of innovation make bioengineering extremely dynamic and this forces researchers to continuous updating. It involves the publication of the results of the latest scientific research. This book covers a wide range of aspects and issues related to advances in bioengineering research with a particular focus on innovative technologies and applications. The book consists of 13 scientific contributions divided in four sections: Materials Science; Biosensors. Electronics and Telemetry; Light Therapy; Computing and Analysis Techniques

VISUALIZATION OF ULTRASOUND INDUCED CAVITATION BUBBLES USING SYNCHROTRON ANALYZER BASED IMAGING

Ultrasound is recognized as the fastest growing medical modality for imaging and therapy. Being noninvasive, painless, portable, X-ray radiation-free and far less expensive than magnetic resonance imaging, ultrasound is widely used in medicine today. Despite these benefits, undesirable bioeffects of high-frequency sound waves have raised concerns; particularly, because ultrasound imaging has become an integral part of prenatal care today and is increasingly used for therapeutic applications. As such, ultrasound bioeffects must be carefully considered to ensure optimal benefits-to-risk ratio. In this context, few studies have been done to explore the physics (i.e. ‘cavitation’) behind the risk factors. One reason may be associated with the challenges in visualization of ultrasound-induced cavitation bubbles in situ. To address this issue, this research aims to develop a synchrotron-based assessment technique to enable visualization and characterization of ultrasound-induced microbubbles in a physiologically relevant medium under standard ultrasound operating conditions. The first objective is to identify a suitable synchrotron X-ray imaging technique for visualization of ultrasound-induced microbubbles in water. Two synchrotron X-ray phase-sensitive imaging techniques, in-line phase contrast imaging (PCI) and analyzer-based imaging (ABI), were evaluated. Results revealed the superiority of the ABI method compared to PCI for visualization of ultrasound-induced microbubbles. The second main objective is to employ the ABI method to assess the effects of ultrasound acoustic frequency and power on visualization and mapping of ultrasound-induced microbubble patterns in water. The time-averaged probability of ultrasound-induced microbubble occurrence along the ultrasound beam propagation in water was determined using the ABI method. Results showed the utility of synchrotron ABI for visualizing cavitation bubbles formed in water by clinical ultrasound systems working at high frequency and output powers as low as used for therapeutic systems. It was demonstrated that the X-ray ABI method has great potential for mapping ultrasound-induced microbubble patterns in a fluidic environment under different ultrasound operating conditions of clinical therapeutic devices. Taken together, this research represents an advance in detection techniques for visualization and mapping of ultrasound-induced microbubble patterns using the synchrotron X-ray ABI method without usage of contrast agents. Findings from this research will pave the road toward the development of a synchrotron-based detection technique for characterization of ultrasound-induced cavitation microbubbles in soft tissues in the future

Submicron gas bubbles in water

Gas bubbles smaller than 1 micrometre in water, commonly referred to as nanobubbles, is a growing field of research and innovation. Applications range from medical imaging and drug delivery to mining industry and environmental remediation. Despite much activity, important questions remain – which are the mechanisms that allow small gas bubbles to be stable against dissolution and are stable nanobubbles really as common and easily generated as is often claimed?This work demonstrates that several common nanobubble generation methods can generate particle agglomerates or oil droplets which can be mistaken for bubbles, whereas stable nano- and microbubbles are less easy to generate than commonly believed. The results further suggest that stable bubbles are normally stable due to a shell of surface-active organic compounds, whereas other proposed stability mechanisms are less likely. An unexpected finding was that sorbitan surfactant stabilized air nanobubbles can form long-lived bubble agglomerates.Holographic Nanoparticle Tracking Analysis (H-NTA) is demonstrated as a powerful new method to detect and differentiate between bubbles and particles in the same dispersion. As H-NTA determines the refractive index of tracked objects, bubbles will differ very significantly from solid particles or oil droplets. The method also enables detection of different populations of particles, agglomerates and oil droplets in the same dispersion

Acoustic Droplet Vaporization: Strategies for Control of Bubble Generation and its Application in Minimally Invasive Surgery.

As a minimally invasive alternative to current cancer treatment, the use of encapsulated, superheated liquid perfluorocarbon droplets has been proposed to treat cancer by occlusion therapy. In response to an acoustic field, these droplets, which are small enough to pass through capillaries, vaporize into large gas bubbles that subsequently lodge in the vasculature. This research investigates strategies to reduce the pressures necessary to achieve acoustic droplet vaporization (ADV), what implications they may have on efficiency, and how the resulting location of bubbles may alter the acoustic field. Two methods to lower the ADV threshold were explored. The first investigated the role of pulse duration on ADV. The second investigated the role of inertial cavitation (IC) external to a droplet by adding ultrasound contrast agent (CA), which has a low IC threshold. At 1.44 MHz, the threshold was found to be 5.5-5.9 MPa peak rarefactional pressure (Pr) for short microsecond pulses and decreased for millisecond pulses to 3.8-4.6 MPa Pr. When CAs were added and long millisecond pulses were used, the ADV threshold decreased to values as low as 0.41 MPa Pr. With the help of CA, the same amount of power was necessary to achieve ADV through an attenuating tissue mimicking (TM) phantom as it was without attenuation and with only droplets. When comparing ADV pressure thresholds, where in situ pressures were used when a TM phantom was present, rarefactional pressure appeared to be the salient determinant. However, careful consideration must be taken when choosing pulse repetition frequencies and amplitude as inertial collapse of both ADV and IC bubbles appears to affect efficient droplet conversion. During in vivo application, treatment planning may be important as backscattering properties of microbubbles created by ADV can augment or obstruct the sound field in the affected area. With strategic targeting and subsequent conversion of droplets into microbubbles, constructive interference due to these effects reduces the transmitted pressures required for proximal ADV, and the attenuation from these bubbles can create a protective boundary for distal areas. The potential result can be a confined area for further ADV where lower pressures are required to cause vaporization.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57723/2/ahlo_1.pd

A multiplexed microfluidic and microscopy study of vasodilation signaling pathways using microbubble and ultrasound therapy

Dans les tumeurs solides, l'hypoxie est un mécanisme de résistance à la radiothérapie bien connu. Il a déjà été démontré que, lorsque les microbulles (MB) sont exposées à une impulsion ultrasonore (US), celles-ci peuvent induire une vasodilatation dans les tissus musculaires. De plus, une impulsion thérapeutique peut être délivrée localement dans la tumeur en dirigeant le faisceau US. Cette approche est donc proposée comme thérapie provasculaire ciblée, guidée par l’imagerie ultrasonore dans les tumeurs afin de réduire l'hypoxie avant la radiothérapie. Le contrôle de la vasodilatation est induit par la production d'oxyde nitrique (NO) par la voie de signalisation cellulaire du eNOS dans les cellules endothéliales. Il a été démontré que l'augmentation de l'ATP extracellulaire active la voie de signalisation du eNOS. Il a aussi été démontré que l’oscillation des MB sous l’effet des US libèrent de l'ATP lorsque le tissu musculaire est traité. Cependant, les effets des différentes conditions ultrasonores et de MB sur la libération d'ATP n'ont pas encore été étudiés. Nous émettons donc l'hypothèse qu'il existe des conditions permettant de maximiser l’activation des voies de signalisation purinergiques (ATP) et d'optimiser leur durée d’activation pour une réponse provasculaire optimale. Les motivations de ce projet sont de tester divers paramètres et d'étudier les interactions MB/cellules dans des conditions d'écoulement, qui sont généralement difficile à mettre en place lorsqu'on utilise des boîtes de Pétri. Pour quantifier plus facilement les voies de signalisation, nous avons créé des puces microfluidiques avec quatre canaux parallèles dans lesquels des cellules ont pu être cultivées. Avec quatre canaux traités lors d’une même impulsion ultrasonore, nous avons aussi augmenté le nombre de données à traiter et nous pouvons observer les effets de plusieurs impulsions lorsque les MB étaient dans un écoulement. En outre, la puce que nous avons développé est capable de donner une concentration en MB différente dans chaque canal afin de pouvoir tester quatre concentrations de MB différente dans des conditions d’écoulement. Les objectifs de ce projet de maîtrise sont donc les suivants : (1) concevoir la puce microfluidique ; (2) être capable de cultiver des cellules dans les canaux microfluidiques ; (3) créer des protocoles pour mesurer la libération d'ATP et la viabilité cellulaire après une impulsion ultrasonore ; (4) observer la capacité de la puce à donner différentes concentrations de MB dans chaque canaux en conditions d’écoulement. Lors de la conception de la puce microfluidique, nous avons créé un environnement dans lequel les quatre canaux de la puce ont des concentrations différentes de microbulles fluides. Ainsi, nous avons atteint les objectifs du projet. Nous avons réussi à introduire dans le canal microfluidique des cellules endothéliales de cordon ombilical humain (HUVEC) et une lignée cellulaire de cancer du sein (4T1). Les monocouches cellulaires créées par chacune des deux lignées cellulaires ont été traitées avec succès par une impulsion thérapeutique ultrasonore lors de l’injection de MB. Nos résultats montrent qu'une augmentation du nombre de cycles et de la pression, libère plus d'ATP et induisent une mortalité cellulaire plus importante. En outre, nous avons établi un lien entre la libération d'ATP et la mortalité cellulaire en comparant différentes impulsions thérapeutiques ultrasonore. Cette analyse a permis de dégager deux tendances. Avec des impulsions à faible énergie, la libération d'ATP est augmenté et on constate une très faible augmentation de la mort cellulaire ; inversement, avec des impulsions à plus forte énergie, la libération d'ATP et la mortalité cellulaire ont augmentés et on atteint un plateau. Ainsi, nos résultats confirment que différents mécanismes de libération d'ATP peuvent être déclenchés par les thérapies MB et US.In solid tumors, hypoxia is a well-known resistance mechanism to radiation therapy. It was previously shown that microbubbles (MBs), when exposed to an ultrasound pulse (US) can cause vasodilation in muscle tissue. Conceptually, the therapeutic pulse can be localized on the tumor by steering the US beam. This approach is therefore proposed as a targeted image-guided provascular therapy in tumors to reduce hypoxia before radiotherapy. However, the effects of US and MB conditions on the relative increase in tumor perfusion remain largely unknown. Vascular control is managed by the production of nitric oxide (NO) through the eNOS pathway inside the endothelial cells. Increases in extracellular ATP have been shown to be a signaling event for the activation of this pathway. Fittingly, MB and US have been shown to release ATP when muscle tissue was treated. However, the effects of therapeutic US and MB parameters on the treatment have not yet been described. We, therefore, hypothesize that there are conditions that will maximize the purinergic signaling pathways (ATP) and optimize their time course for an optimal provascular response. The motivation for this project came from the desire to test various parameters and study MB/cell interactions in flowing conditions, which are typically limited when using petri dish setups. To quantify more easily the signaling pathways, we created microfluidic chips with four parallel cell coated channels. This chip allowed us to increase the throughput when using a single US exposure in static conditions and with the ability to support multiple US exposures with MB replenishment in flowing conditions. Also, the custom-made chip multiplexes the bubble concentration to obtain four channels with different flowing microbubble concentrations. The goals of this master’s project were thus: (1) to design the microfluidic chip; (2) to demonstrate the capacity for cell culture; (3) create protocols for measuring ATP and cell viability after therapeutic pulses; (4) to demonstrate repeatable flowing conditions with the multiplexed MB concentration. On the design of the microfluidic chip, we were successful at creating an environment where four of the four channels in the chip have different concentrations of flowing microbubbles. Thus, fulfilling the project's goals. We have succeeded in seeding both Human Umbilical Vein Endothelial Cells (HUVECs) and a breast cancer cell line (4T1) into the microfluidic channel. The cell monolayers created by both cell lines were successfully treated with an US and MB therapeutic pulse. Our results support that an increase in both, cycles and pressure, release more ATP and cause more cell death. Further, we linked ATP release to cell death by comparing different therapeutic pulses. From this analysis, two trends appeared. With lower energy pulses, ATP release increased sharply with a very small increase in cell death; conversely, with higher energy pulses, ATP release continued to increase with cell death but reached a plateau. Thus, our results support that different mechanisms of ATP release can likely be triggered by MB and US therapy

Nanobubbles in water - how to identify them and why they are stable

Gas bubbles smaller than 1 micrometer in water, commonly referred to as nanobubbles, is a growing field of research and innovation. Applications range from medical imaging and drug delivery to mining industry and environmental remediation. There are many possibilities but important questions remain – how is it possible for small gas bubbles to be stable against dissolution and how can they be detected and differentiated from solid particles and oil droplets ?In this work we demonstrate that several common nanobubble generation methods can generate contamination particles which can be mistaken for bubbles and that with sufficient cleanliness, neither particles, droplets or bubbles are generated. Theories on nanobubble stability that does not include impurities can thus be dismissed. (Paper 1). Lipid stabilization and the dynamic equilibrium model based on hydrophobic dirt particles appear to be the only valid models for nanobubble stability at present.We furthermore demonstrate Holographic Nanoparticle Tracking Analysis (H-NTA) as a powerful new method to detect and differentiate between nanobubbles and nanoparticles in the same solution (Paper 2). As H-NTA determines the refractive index of tracked objects, bubbles will differ very significantly from solid particles or oil droplets. The refractive index of a bubble also indicates the amount of adsorbed material as well as possible clustering of multiple bubbles. The method also powerfully enables detection of different particle populationsclose in size and refractive index in a dispersion. The size range is 0.3-0.4 mm to 1.5 mm