Disorders of the central nervous system (CNS) contribute almost 800 billion euros in annual European healthcare costs. New compounds, effective in animal models, hardly work in humans, mostly due to the inability to cross the Brain Blood Barrier (BBB). In these conditions cost-effective tools to alter BBB and, more generally, endothelial layer permeability is desirable before proceeding to expensive and time-consuming animal studies. INVICTUS (IN VItro Cavitation Through UltraSound) originates within the ERC-AdG project Bubbles from Inception to Collapse (BIC) and concerns the development of a biomimetic microfluidic platform to be made turnkey available to biologists, clinicians and pharmacologists. Its potential is significant, given the well known societal and economical impact of degenerative diseases, the enormous investment and the long times of pre-clinical trials. Limiting/avoiding animal experimentation has an evident ethical impact and is associated with substantial economic savings and organisational simplification.
INVICTUS is conceived to guarantee the integration of a vessel-on-a-chip with a complete lumen of endothelial cells and the use of controlled cavitation induced by ultrasound. Already pursed in in vivo animal models, this approach is extended here to a high-fidelity, in vitro biomimetic device that will bring to market new crucial features such as the three-dimensional geometry of realistic-size vascular channels featuring an actual endothelial barrier, the correct perfusion rate, the appropriate physiological shear stress exerted on the endothelial cells and the ability to reproduce biochemical interactions between different, healthy and diseased, tissues.
The platform is designed to be housed on the stage of a microscope ensuring the correct working distance of the objectives and allowing real-time measurements. The vessel-on-a-chip is locked at the center of the bottom side of the platform, where the optical access is available. The geometry of INVICTUS allows the positioning of a piezo transducer with the correct distance to have proper ultrasound irradiation and the immersion of a thermal controller. To induce the mechanical effects of cavitation, Sonovue® microbubbles, typically used as contrast agent in US imaging, are injected in the vascular channels. When ultrasound radiation forces are applied, cavitation occurs producing microstreaming and micro jets acting on the inter-endothelial junctions leading to reversible permeability enhancement.
To validate the proof of concept, the INVICTUS platform has been exploited to investigate the effect of ultrasound-excited microbubbles on endothelial integrity. We have demonstrated that the proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications. The results show that the mechanical effects of ultrasound-excited microbubbles lead to the formation of inter-endothelial gaps that cause barrier permeabilisation. A relevant increase of the total inter-cellular gap area has been observed with respect to an untreated vascular channel. Interestingly, the effect of bubble-mediated US radiation was found to be transient and reversible, since the endothelial barrier was shown to revert to the original state after irradiation.
The significant results obtained during INVICTUS development and validation, confirm the validity of this innovative methodology for the quantitive understanding of cavitation assisted drug delivery.
