Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3D cell culture, perfusion flow, and membranefree soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh speed camera (17 Mfps) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated d

The role of ultrasound-driven microbubble dynamics in drug delivery : from microbubble fundamentals to clinical translation

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future

Perspectives on cavitation enhanced endothelial layer permeability

Traditional drug delivery systems, where pharmaceutical agents are conveyed to the target tissue through the blood circulation, suffer of poor therapeutic efficiency and limited selectivity largely due to the low permeability of the highly specialised biological interface represented by the endothelial layer. Examples concern cancer therapeutics or degenerative disorders where drug delivery is inhibited by the blood-brain barrier (BBB). Microbubbles injected into the bloodstream undergo volume oscillations under localised ultrasound irradiation and possibly collapse near the site of interest, with no effect on the rest of the endothelium. The resulting mechanical action induces a transient increase of the inter-cellular spaces and facilitates drug extravasation. This approach, already pursed in in vivo animal models, is extremely expensive and time-consuming. On the other hand in vitro studies using different kinds of microfluidic networks are firmly established in the pharmaceutical industry for drug delivery testing. The combination of the in vitro approach with ultrasound used to control microbubbles oscillations is expected to provide crucial information for developing cavitation enhanced drug delivery protocols and for screening the properties of the biological interface in presence of healthy or diseased tissues. Purpose of the present review is providing the state of the art in this rapidly growing field where cavitation is exploited as a viable technology to transiently modify the permeability of the biological interface. After describing current in vivo studies, particular emphasis will be placed on illustrating characteristics of micro-devices, biological functionalisation, properties of the artificial endothelium and ultrasound irradiation techniques

Increasing the Endothelial Layer Permeability Through Ultrasound-Activated Microbubbles

Drug delivery to a diseased tissue will be more efficient if the vascular endothelial permeability is increased. Recent studies have shown that the permeability of single cell membranes is increased by ultrasound in combination with contrast agents. It is not known whether this combination can also increase the permeability of an endothelial layer in the absence of cell damage. To investigate the feasibility of controlled increased endothelial layer permeability, we treated monolayers of human umbilical vein endothelial cells with ultrasound and the contrast agent BR14. Barrier function was assessed by measuring transendothelial electrical resistance (TEER). Ultrasound-activated BR14 significantly decreased TEER by 40.3% ?? 3.7% (p < 0.01). After treatment, no cell detachment or damage was observed. In conclusion, ultrasound-activated BR14 microbubbles increased the endothelial layer permeability. This feature can be used for future ultrasound-guided drug delivery system

Investigation Of Ultrasound Targeted Microbubbles As A Therapeutic Gene Delivery System For Prostate Cancer

A major challenge for effective gene therapy is systemic delivery of viruses carrying therapeutic genes into affected tissue. The immunogenic nature of human adenoviruses (Ads) limits their use for intratumoral (IT) injection in gene therapy. Ads transfection is further hampered by the fluctuating presence of Coxsackie and Adenovirus Receptor (CAR) and integrins on the cells’ surface. To circumvent these limitations we developed a novel approach wherein Ads are encapsulated inside the shell of lyophilized, lipid-encapsulated, perfluorocarbon microbubbles (MBs)/ultrasound (US) contrast agents, which act as delivery vehicles for a sitespecific gene transfer system. We performed infection studies with Ad.GFP (Green Fluorescent Protein), Ad.mda-7 (melanoma differentiation associated gene 7) and CTV.mda-7 on human DU145 and mouse prostate cancer cells as well as observed enhanced GFP expression when Ad.GFP was delivered by MBs and US. Our results show that US breaks open the MB/Ads complexes by undergoing cavitation at the sonoporated site, which allows Ads to transfer their transgene only to the sonoporated region. Cavitation collapse of the MBs creates small shockwaves that increase cell permeability by forming temporary micropores on the cell surface bypassing the receptormediated dependence of Ads for transfection. Fetal bovine serum (FBS) containing complement did not allow the unprotected Ads to infect the cells; however, MBs complexed with Ad.GFP did infect DU145 and TRAMP-C2 cells in a FBS rich media. We studied MB assisted gene delivery of reporter (GFP) and therapeutic genes (p53, Rb, Rb2 (p130) and Mda-7/IL-24) into prostate cancer (PC) xenografted in immune-compromised athymic mice. The results demonstrated that MBs protect the host from unspecific viral immune response thus protecting the viral payload and allowing for intravenous (IV) injection rather than IT injection. Additionally, Ad gene transfer was enhanced at the targeted/sonoporated mice tumor xenografts. This research demonstrated mda-7’s efficacy in reducing primary (treated) and untreated tumors that simulated the presence of metastasis in athymic mice xenograft models bearing human PC cells. Bystander anti-tumor activity of mda-7, a secreted cytokine was noted for non-targeted tumors. Earlier in vitro studies on the combination of radiation and gene therapy (Ad.p53, Ad.Rb, and Ad.p130) demonstrated an increase in the percentage of cell death for DU145 cells. We also studied UTMD (ultrasound targeted microbubble destruction) gene therapy in combination with external beam radiation for radiation resistant PC. The results demonstrated an enhanced therapeutic benefit of tumor suppressor genes in radiation resistant PC. We also demonstrated an increase in the expression of tumor suppressor genes at the tumor site due to MBs and US. These findings highlight the potential therapeutic benefit of this novel image guided gene transfer technology alone or in combination with external beam radiation for prostate cancer patients with therapy resistant disease

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The Physical Mechanism of Blood-Brain Barrier Opening Using Focused Ultrasound and Microbubbles

The key to effective treatment of neurological diseases resides in the safe opening of the blood-brain barrier (BBB), a specialized structure that impedes the delivery of therapeutic agents to the parenchyma. Despite the fact that several approaches have been successful in overcoming the BBB impermeability, none of them can induce localized BBB opening noninvasively except for focused ultrasound (FUS) in conjunction with microbubbles. The physical mechanism behind the opening, however, has not been identified. Insight into the mechanism can be critical for delineating the safety profile for in both small and large animals alike. Therefore the purpose of this dissertation is to first determine the physical mechanism of FUS-induced BBB opening in mice and then translate this approach to non-human primates. To accomplish this goal, an in vivo transcranial cavitation detection system was developed and tested, built in phantoms and in vivo, to monitor the behavior of the microbubbles in the FUS bean, and to determine the type of cavitation, i.e., the activation of bubbles in an acoustic field, during BBB opening. We showed that the inertial cavitation (IC), a collapse of a bubble, which can vary from a fragmentation of the bubble to shock wave and liquid jets depending on the pressure, thereby damaging the endothelial cells of the brain capillaries, was not required to induce BBB opening in mice. With this system, the role of microbubble properties, including the diameter and shell components, in the BBB opening were determined. When the BBB opens with stable cavitation (SC), i.e., relatively moderate amplitude changes in the bubble size, the bubble diameter is similar to the capillary diameter (i.e., at 4-5, 6-8 µm) while with inertial cavitation it is not (i.e., at 1-2 µm). The bubble may thus have to be in closer proximity to the capillary wall to induce BBB opening without IC. The BBB opening properties, such as volume and permeability, however, were not affected by the shell component of the microbubbles in mice. The connection between the physical and physiological mechanism was then investigated to identify the lowest peak rarefactional pressure BBB opening threshold at 1.5 MHz (0.18 MPa). A sufficiently long pulse (pulse length = 0.5 ms) was required for the SC to induce BBB opening at the lowest pressure. However, the tight junctions, the main formation of the BBB, were found not to be disrupted after sonication at both low (0.18 MPa) and high (0.45 MPa) pressures. Therefore, the transcellular pathway may be the main route of the FUS-induced BBB opening. Finally, the cavitation-guided BBB opening system was used to induce reversible BBB opening in non-human primates. This is a major step towards clinical feasibility. In conclusion, a transcranial cavitation detection system was developed, in order to characterize the physical mechanism, the role of the microbubbles, and the corresponding physiological response of the FUS-induced BBB opening

Ultrasound Contrast Agents and Their Use in Triggered Drug Delivery

Ultrasound imaging is non-ionizing radiation and an inexpensive, non-invasive diagnostic tool. However, ultrasound alone results in poor image resolution as well as shallow depth of view and is therefore insufficient for many diagnostic processes. In order to improve the resolution of the image, a contrast agent is injected into the bloodstream, but with this comes an added safety risk as well as complications due to a relatively short lifespan. To improve currently available contrast agents, a novel design is formulated. This contrast agent is comprised of lipid microbubbles within the aqueous core of polymer shell microcapsules, termed nested microbubbles. Nested microbubbles also function as prospective ultrasound triggered drug delivery vehicles when the polymer shell is replaced with a lipid bilayer and the core now contains an aqueous drug in addition to the microbubbles. This formulation has dual benefits: drug release from the membrane when microbubbles are present within the liposome and an increase in image brightness during ultrasound guided procedures. One important aspect of this research is the relationship between the type of cavitation and resulting membrane disruption. Two regimes exist during the membrane damage-inducing events caused by cavitating microbubbles which can allow for tailored drug release behavior at the desired location within the body.Ph.D., Chemical Engineering -- Drexel University, 201