Cavitation-enhanced permeability in a vessel on a chip

Methods combining ultrasound and microbubbles (USMB) offer the unique capability of non-invasively, locally and transiently increase endothelial permeability [1]. This is crucial for the delivery of pharmaceutical agents, injected into the blood circulation, since the real efficiency of a therapy depends on the rate and ability of a macromolecules to cross the endothelial barrier and reach the intended target. Molecule passage through this biological barrier is hampered by the endothelium, lining the innermost surface of blood vessels, consisting of a continuum layer of specialized cells close together to form a size-selective membrane. In this contest, cavitation-assisted permeation shows promise for reversibly altering the barrier integrity, opening gaps between endothelial cells and doing so facilitating the diffusion of pharmaceutical agents out of vessel. Although acoustic cavitation is already exploited in in vivo animal models for drug delivery testing, the in vitro approach offers the possibility to obtain well-controlled procedures, saving in cost and time [2]. Here, a platform integrating in vitro blood vessels and acoustic cavitation is used to test the feasibility of micro bubbles (MBs) cavitation-enhanced endothelial permeability. We induce MBs (Sonovue® contrast agent) stable cavitation, evoked by low-intensity ultrasound exposure (Mechanical Index (MI) = 0.4, 0.72), in a microfluidic device purposely designed [3] to mimic micro-blood vessel. The bio-inspired device consists in a PDMS microfluidic network with a central circular tissue compartment enclosed by two independent vascular channels mimicking the three-dimensional morphology, size and flow characteristics of a micro vessel in vivo. The device is previously cultured with Human Umbilical Vein Endothelial Cells (HUVECs) with a reliable and reproducible protocol [4] that allows endothelial cells to form a complete lumen under physiological shear stresses. Immunofluorescence microscopy is then exploited in order to monitor vascular integrity following vascular endothelial cadherin (VE-Cadherin), the most determinant protein for vascular permeability. The endothelial membrane permeability is evaluated through a dedicated optical/acoustic set-up in presence of ultrasound-activated MBs driven by 1 MHz-unfocused transducer. The basic set up is designed and adapted to host the bio-inspired device, the piezoelectric transducers within a water-filled and temperature-controlled costume chamber located on the microscope stage. Measurements of fluorescent dye diffusion towards the biological membrane has been carried out with a time lapse acquisition under a confocal microscope operated in epifluorescence mode. An image analysis on the intensity change due to fluorescence accumulation in the tissue compartment is performed to obtain quantification of permeability. Intercellular gaps were firstly identified by inspection using ImageJ software and then post-processed in order to increase the contrast and binarize the image using a threshold method with the same cut-off value for all Regions of Interest. The gap area was then quantified counting the black pixels of the central connected blob in each binarized image. The results show that MBs amplify the ultrasound effect, leading to the formation of inter-endothelial gaps, proportionally to the applied acoustic pressure, and causing barrier permeabilization. Moreover, endothelium recovery was completely achieved after 45 minutes from the USMB exposure with gap area distribution returning to the control levels. To conclude, the proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability providing a novel methodology for the quantitative understanding of cavitation assisted drug delivery. [1] K. Kooiman, H. J. Vos, M. Versluis, and N. de Jong, “Acoustic behaviour of microbubbles and implications for drug delivery,” Advanced drug delivery reviews, vol. 72, pp. 28–48, 2014. [2] Peruzzi, G. Perspective on cavitation enhanced endothelial layer permeabiliry, Colloids and surface B: biointerfaces 168 (2018), 3-93 [3] S.P.Deosarkar, et al. A novel dynamic neonatal blood-brain barrier on a chip, Plos One, 10(11) (2015), p. e0142725