Targeted intracellular delivery is a goal of many novel drug delivery systems to treat site-specific diseases thereby increasing the effectiveness of drugs and reducing side effects associated with current drug administration. The development of ultrasound-enhanced delivery is aimed at providing a targeted means to deliver drugs and genes intracellularly by utilizing ultrasound s ability to non-invasively focus energy into the body and generate cavitation, which has been found to cause transient poration of cells. To address some of the current issues in this field, the goals of this study were (i) to develop a measurement of cavitation to correlate with cellular bioeffects and (ii) to evaluate the ability of ultrasound to target delivery into cells in viable tissue. In addition, this study sought to exploit the shear-based mechanism of cavitation by (iii) developing a simplified device to expose cells to shear stress and cause intracellular uptake of molecules. This study has shown that broadband noise levels of frequency spectra processed from cavitation sound emissions can be used to quantify the kinetic activity of cavitation and provide a unifying parameter to correlate with the cellular bioeffects. We further demonstrated that ultrasound can target delivery of molecules into endothelial and smooth muscle cells in viable arterial tissue and determined approximate acoustic energies relevant to drug delivery applications. Lastly, we developed a novel device to expose cells to high-magnitude shear stress for short durations by using microfluidics and demonstrated the ability of this method to cause delivery of small and macromolecules into cells. In conclusion, this work has advanced the field of ultrasound-enhanced delivery in two major areas: (i) developing a real-time non-invasive measurement to correlate with intracellular uptake and viability that can be used as means to predict and control bioeffects in the lab and potentially the clinic and (ii) quantitatively evaluating the intracellular uptake into viable cells in tissue due to ultrasound that suggest applications to treat cardiovascular diseases and dysfunctions. Finally, by using shear forces generated in microchannels, we have fabricated a simple and inexpensive device to cause intracellular uptake of small and large molecules, which may have applications in biotechnology.

Capacitive Micromachined Ultrasonic Transducers (CMUTs) have been introduced as a viable alternative to piezoelectric transducers in medical ultrasound imaging in the last decade. CMUTs are especially suitable for applications requiring small size such as catheter based cardiovascular applications. Despite these advantages and their broad bandwidth, earlier studies indicated that the overall sensitivity of CMUTs need to be improved to match piezoelectric transducers. This dissertation addresses this issue by introducing the dual-electrode CMUT concept. Dual electrode configuration takes advantage of leveraged bending in electrostatic actuators to increase both the pressure output and receive sensitivity of the CMUTs. Static and dynamic finite element based models are developed to model the behavior of dual-electrode CMUTs. The devices are then successfully fabricated and characterized. Experiments illustrate that the pulse echo performance is increased by more than 15dB with dual-electrode CMUTs as compared to single electrode conventional CMUT. Further device optimization is explored via membrane shape adjustment by adding a center mass to the design. Electromechanical coupling coefficient (kc2) is investigated as a figure of merit to evaluate performance improvement with non-uniform/uniform membrane dual-electrode CMUTs. When the center mass is added to the design, the optimized non-uniform membrane increases the electromechanical coupling coefficient from 0.24 to 0.85 while increasing one-way 3dB fractional bandwidth from 80% to 140% and reducing the DC bias requirement from 160V to 132V. The results of this modeling study are successfully verified by experiments. With this membrane shape adjustment, significant performance improvement (nearly 20dB) is achieved with the dual-electrode CMUT structure that enables the CMUT performance to exceed that of piezoelectric transducers for many applications.

We used megahertz pulsed ultrasound and studied gene transfection with a human prostate cancer cell line. We first studied the compromise of cell viability and uptake efficiency and found out that increasing sonication temperature or changing US contrast agents could improve drug/gene delivery mediated by US exposure. We also found that accounting for cell debris after sonication was important to correctly determine cell viability. Next, we verified the capability of US to deliver DNA into the cell nuclei, which is necessary for successful gene transfection. Under the optimal sonication conditions, ~ 30% of cells showed DNA uptake right after US exposure and most had a portion of DNA already localized in the cell nuclei. The maximum transfection efficiency was ~ 12% at 8 h post US exposure. From the DNA perspective, ~ 30% of DNA was localized in the cell nuclei immediately after US exposure and ~ 30% was in the autophagosomes/ autophagolysosomes with the rest ¡°free¡± in the cytoplasm. At later time up to 24 h, DNA continued to be distributed ~ 30% in the nuclei and most or all of the rest in autophagosomes/autophagolysosomes. Our results showed that US was able to deliver DNA into the cell nuclei shortly after the treatment and that the rest of DNA was mostly cleared by autophagosomes/autophagolysosomes. To further increase transfection efficiency, we then studied the differences between live cells with DNA uptake and those with successful gene transfection post US exposure using cell sorting, cell cycle and microarray analysis. Cells with gene transfection were found to accumulate at the G1 phase of cell cycle and associate with the up-regulation of 32 genes (e.g., GADD45¦Á) and the down-regulation of 46 genes (e.g., TOP2¦Á). Drugs that regulate the expression levels of GADD45¦Á and TOP2¦Á were found to further enhance the transfection mediated by US. A maximun increase of ~ 2 fold in transfection efficiency was observed when cells were sonicated with 0.6 mg/mL ethyl methanesulfonate to up-regulate GADD45¦Á. These results suggestted that using drugs that regulate certain introcellular processes could further enhance US-mediated gene transfection. Over a broad range of US conditions, the integrity of three common gene delivery vectors, plasmid DNA, siRNA and adeno-associated virus, were not affected by US exposure. This thesis verified that US was able to delivery DNA into the cell nuclei to facilitate rapid gene transfection, and provided a proof of princible that by modulating certain intracellular processes, the efficiency of US-mediated gene transfection could be further increased. US could potentially be a safe and efficient method for gene therapy.

Thesis (M. S.)--Chemical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Prausnitz, Mark; Committee Member: Bommarius, Andreas; Committee Member: Jones, Christopher; Committee Member: Sambanis, Athanassios. Part of the SMARTech Electronic Thesis and Dissertation Collection.

No description available.

Ultrasound has been identified as a possible non-invasive drug delivery device that could avoid many of the problems found in traditional therapeutics. Studies have shown that ultrasound can deliver molecules into cells; however, the applicability of ultrasound has been limited due to uncontrollable cellular viability losses after sonication. In this study, we sought to quantify the heterogeneous bioeffects of ultrasound in order to gain more insight into how ultrasound affects cells. We were also concerned with identifying the causes of and preventing programmed cell death caused by ultrasound exposure. In order to accomplish these objectives, we used flow cytometry to group cells into quantifiable characteristic populations. This allowed us to identify the relative importance of different forms of rapid cell death. We found that up to 65% of cells (at the highest ultrasound pressure studied) can lose viability rapidly and, for the first time, quantified them among three distinct populations: (1) cells that retain normal size but lose plasma membrane integrity; (2) intact nuclei surrounded by plasma membrane remnants; (3) debris resulting from cellular lysis. Our analysis was supported by mechanical sorting of these populations and subsequent imaging using confocal microscopy. We then monitored the viable populations for 6 h after ultrasound exposure. Results indicated that up to 15% of viable cells (at the highest ultrasound pressure studied) underwent apoptosis, which we showed was associated with an influx of intracellular Ca2+; therefore, we developed a method of chelating intracellular Ca2+ after sonication in an effort to maintain viability of those cells. Using this technique, we showed for the first time that cells could be saved, and we were able to prevent apoptosis by 50%, thereby increasing the overall viability of cells exposed to ultrasound. We conclude that ultrasound is a useful method to deliver molecules into cells and that appropriate selection of sonication conditions can minimize cell death by rapid and apoptotic mechanisms.

Thesis (MTech (Electrical Engineering))--Cape Peninsula University of Technology, 2016. / Ultrasound is widely used in most medical clinics, especially obstetrical clinics. It is a way of imaging methods that has important diagnostic value. Although useful in many different applications, diagnostic ultrasound is especially useful in antenatal (before delivery) diagnosis. The use of two-dimensional ultrasound (2DUS) in obstetrics has been established. However, there are many disadvantages of 2DUS imaging. Several researchers have published information on the significance of patients being shown the ultrasound screen during examination, especially during three- and four-dimensional (3D/4D) scanning. In addition, a form of ultrasound, called keepsake or entertainment ultrasound, has boomed, particularly in the United States. However, long-term epidemiological studies have failed to show the adverse effects of ultrasound in human tissues. Until now, there is no proof that diagnostic ultrasound causes harm in a human body or the developing foetus when used correctly. While ultrasound is supposed to be absolutely safe, it is a form of energy and, as such, has effects on tissues it traverses (bio-effects). The two most important mechanisms for effects are thermal and non-thermal. These two mechanisms are indicated on the screen of ultrasound devices by two indices: The thermal index (TI) and the mechanical index (MI). These are the purposes of this thesis: • evaluate end-users’ knowledge regarding the safety of ultrasound; • evaluate and make a comparison between acoustic output indices (AOI) in B-mode (2D) and three-dimensional (3D) ultrasound – those measured by thermal (TI) and mechanical (MI) indices; • assess the acoustic output indices (AOI) to benchmark current practice with a survey conducted by the British Medical Ultrasound Society (BMUS); and • review how to design 2D and 3D arrays for medical ultrasound imaging

M.Tech. (Chiropractic) / The aim of the study was to compare the efficacy of treating the active levator scapulae trigger point (TP1) with either chiropractic adjustments combined with ischemic compression or chiropractic adjustments combined with ultrasound therapy in physically active people in order to determine which of the two treatment protocols was superior.This study was a comparative study consisting of two groups of fifteen participants each. Participants were between the ages of eighteen and forty-five and there was an equal male to female ratio. Prior to becoming a participant in this study individuals were assessed according to the inclusion and exclusion criteria. The International Physical Activity Questionnaire, a clinical case history, full physical examination, a cervical regional examination and examination of the levator scapulae muscle for an active central trigger point (TP1) were completed. The method of treatment for each participant was determined by random group allocation. Group 1 received cervical spine chiropractic adjustments combined with ischemic compression to the active levator scapulae trigger point. Group 2 received cervical spine chiropractic adjustments combined with ultrasound therapy to the active levator scapulae trigger point. Subjective and objective readings were based on the above treatment protocols.Treatment consisted of seven consultation sessions over a three week period. There were six treatment visits with the seventh visit used only for data collection. There were two treatments each week with at least two days in between visits. The third week consisted of three visits with the last visit used only for data collection. Subjective data was collected from the Vernon-Mior Neck Pain and Disability Index Questionnaire and the Numerical Pain Rating Scale. Objective data was collected from the pressure algometer readings. Subjective and objective data was collected before treatment on the first and fourth visits and on the seventh final data collection visit. Analysis of the data collected was done by a statistician. The chiropractic adjustments used were based on motion palpation findings on the treatment visits and re-assessed on each visit.Clinically significant improvements regarding neck pain and disability and trigger point severity were seen in both Group 1 and Group 2 over the three week period. Group 2 showed greater improvements in all subjective and objective readings over the three weeks compared to Group 1.

Over the last six decades, the field of drug delivery has advanced considerably, from sustained oral release technology to pH-responsive polymers. Innovation in the space has progressed alongside the development of new categories of drugs, as well as improvements in electronics and material science which have enabled new modalities of external stimulation. Nevertheless, the traditional challenges of drug delivery persist, including the need to reduce off-target toxicity, minimize invasiveness of administration, and bypass biological barriers; these challenges are particularly apparent for drug delivery applications in difficult-to-reach areas of the body, such as tumors or areas beyond the blood-brain barrier. Furthermore, as therapeutics become more targeted, the need for corresponding delivery methods becomes even more vital to ensure treatment effectiveness with minimal side effects. In this dissertation, we aim to demonstrate a new strategy for on-demand and localized drug delivery which is easy to fabricate and delivers a large payload relative to device size, is responsive to external stimulation for triggered release, and can be integrated into a system for real-time actuation during a physiological process. In Aim 1, we developed a microfluidic fabrication technique for making biphasic microcapsules loaded with model drug. This method relied on microfluidic droplet methods, with sufficient interfacial tension between two on-chip phases to cause droplet formation. Typically, these systems rely on an aqueous-oil interface for sufficient interfacial tension; to fabricate a biocompatible microcapsule, we formed biphasic microcapsules composed of an aqueous-based inner and outer phase, without an oil intermediate phase, with aqueous two-phase system properties. Additionally, we incorporated on-chip photopolymerization, designing the microfluidic chip and light source to minimize refracted ultraviolet exposure. The resulting drug-loaded microcapsules were stable, with minimal background leakage. This fabrication technique can produce a high-throughput supply of monodisperse microcapsules, which can be modified for a variety of therapeutic payloads and easily injected in targeted region in the body. In Aim 2, we adapted these drug-loaded microcapsules for ultrasound-triggered release. Focused ultrasound (FUS) is a minimally-invasive method of stimulating release from a device, which can penetrate deep within the body and is compatible with a variety of materials; when applied at sufficient intensity and duration, it can induce heating, cavitation, or both. We tuned the applied ultrasound parameters to minimize temperature increases in surrounding tissue phantoms, while inducing step-like release profiles from the microcapsules over the course of multiple cycles of pulsed FUS. Under these applied conditions, we detected acoustic signatures consistent with inertial cavitation and visually observed structural breakdown of the microcapsules corresponding to cavitation-related effects. This release strategy is highly targeted, inducing drug release from microcapsules within a narrow focal area with minimal risk to surrounding tissue. Finally, in Aim 3, we performed in vitro demonstrations of drug-loaded actuators, as initial demonstrations towards a system of integrated sensors, actuators, and adaptive learning algorithms for closed-loop control over physiological processes involved in wound healing. We experimented with both the aforementioned microcapsules and with a liposome-loaded scaffold as drug-loaded actuators, and tested both actuators with three ultrasound transducers which offered a range of portability, intensity ranges, and imaging capacities. Next, we developed in vitro testing setups incorporating the actuators with either a cell monolayer or a three-dimensional cell construct, mimicking a wound site, and validated ultrasound-triggered drug-release with minimal cell damage. To demonstrate cell uptake of the released therapeutic agents, we modified the microcapsules’ payload, performed the in vitro release experiments, and then observed correlating cell response over the following week of culturing. These demonstrations have provided guidance towards a more integrated system, which will validate the impact of the localized actuators in stimulating enhancing wound healing rates. More broadly, the eventual integrated system, incorporating both sensors and the adaptive algorithm, will be able to sense and respond to physiological changes within a wound in real-time. This work explores how wireless, deep-tissue devices coupled with external control modalities will facilitate interventions with high spatiotemporal accuracy; when combined with sensing and regulating algorithms, it will empower real-time monitoring and interventions in physiological processes. Aim 1 focused on the fabrication of such implantable microcapsule devices and Aim 2 demonstrated a method for triggering the devices using an external control modality. In Aim 3, we investigated a use case for these microcapsules to promote rapid wound healing, alongside flexible electronics, sensors, and additional actuators. To provide additional context on implantable microdevices and biocompatibility, we provide a framework for designing medical microrobotics in Appendix I and an application of a thermally-responsive hydrogel coating in Appendix II. Overall, the sum of this work illustrates the potential impact of soft microdevices for localized and on-demand applications, towards a future of spatiotemporally-targeted biological interventions.

In addition to protecting the brain from circulating pathogens and neurotoxins, the blood-brain barrier (BBB) limits both the delivery of drugs to the brain and the migration of neurological disease biomarkers from the brain into the blood. Focused-ultrasound blood-brain barrier opening (FUS-BBBO) addresses both of these transport limitations by transiently and noninvasively opening the BBB. Although originally designed as a drug delivery method, FUS-BBBO has also been shown to be an effective neuroimmunotherapy and method of improving liquid biopsy specificity for neurological disease. Prior to the work presented herein, the mechanism of FUS-BBBO neuroimmunotherapy remained poorly characterized and FUS-BBBO liquid biopsy remained poorly optimized. Initially, we present the temporal response of brain macrophages to FUS-BBBO. Due totheir role as the main phagocyte in the brain and the well-documented association between their dysfunction and neurodegenerative disease progression, we hypothesized that FUS-BBBO affects brain macrophage population composition and phenotype. Utilizing temporal single-cell RNA sequencing, we establish that treatment remodels the immune landscape via a number of processes including microglia proliferation, disease-associated microglia population size increase, and central-nervous-system associated macrophage recruitment. To further elucidate the functional role of the brain macrophage response to FUS-BBBO, we find that their depletion is associated with significantly decelerated BBB restoration. Secondly, we compare FUS-BBBO with two other methods of focused ultrasound neuroimmunotherapy, focused ultrasound neuromodulation (FUS-N) and focused ultrasound with microbubbles without BBBO (FUS+MB). FUS-N utilizes FUS parameters that alter neuronal connectivity via a combination of mechanosensitive receptor interactions and transient hypothermia without the injection of microbubbles (MB). FUS+MB is the combination of MB and FUS below the pressure threshold for BBBO (FUS+MB). FUS+MB has been shown to trigger morphological activation of brain macrophages and has proven efficacious as a method of immunotherapy within the peripheral nervous system. Due to the findings of brain macrophage modulation in response to FUS-BBBO, we compare brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s Disease (AD) pathology. We identify FUS-BBBO as the paradigm which maximizes brain macrophage modulation including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between FUS cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to characterize the effect of FUS-BBBO on brain macrophage distribution in both wild-type and Alzheimer’s disease animals. Given their relevance within neurodegeneration and perturbation response, we emphasize the distribution of three brain macrophage populations - disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find a genotype-specific redistribution of each population, with an overall trend towards increased interaction with the brain-cerebrospinal fluid barrier after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. Finally, we investigate the role of extracellular vesicles (EVs) in both the mechanism ofFUS-BBBO neuroimmunotherapy and as a method of improving FUS-BBBO liquid biopsy. EVs are lipid vesicles that are responsible for the transport and exchange of diverse cargo between cells and have been reported to modulate the immune system. Isolation of EVs has emerged as a method of improving biomarker detection. Prior to this study, the effect of FUS-BBBO neuroimmunotherapy on EV concentration and content remained unexplored. We investigate the concentration and content of isolated EVs from the serum of mice and Alzheimer’s Disease patients prior to and after treatment with FUS-BBBO. We illustrate a 100% increase in EV concentration one hour after treatment in both mice and patients. Furthermore, we illustrate an increase in murine EV RNA that is associated with the previously reported neuroimmunotherapeutic responses to FUS-BBBO including synaptic remodeling and neurogenesis. Finally, we illustrate an increase in AD biomarker concentration within the patient EVs three days after treatment that is proportional to the volume of blood-brain barrier opening. Overall, we establish that FUS-BBBO drug-free neuroimmunotherapy triggers complex brain macrophage modulation in a manner incomparable by other FUS neuroimmunotherapy paradigms. Furthermore, we illustrate the effect of FUS-BBBO on EV concentration and content in both preclinical and clinical experiments, indicating the role of EVs in FUS-BBBO neuroimmunotherapy and their utility as a method of improving liquid biopsy specificity. The results presented herein support the potential of FUS-BBBO as both a method of neuroimmunotherapy and a method of amplifying liquid biopsy specificity in Alzheimer’s Disease.