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. e014272

Non Linear Ultrasound Doppler and the Detection of Targeted Contrast Agents

One of the main challenges in molecular imaging with targeted contrast agents is the detection and discrimination of attached agents from the rest of the signals originating from freely flowing agents and tissue. The aim of this thesis was to develop methods for the detection of targeted microbubbles. One approach consisted of investigating the use of nonlinear Doppler for this purpose. Nonlinear Doppler enables the differentiation of moving from non-moving and linear from nonlinear scattering. Targeted microbubbles are static and nonlinear scatterers and they should be detected using this technique. A novel nonlinear Doppler technique: Pulse subtraction Doppler, was developed and compared to pulse inversion Doppler. It is shown that both techniques lead to similar Doppler spectra and depending on the medical applications and the equipment limitations, both techniques have benefits. This served as a starting point for the derivation of a generalised nonlinear Doppler technique, based on combined linear pulse pair sequences and tested in a simulation study. The response from a single microbubble was simulated for different pulse combinations and the pulse sequences were compared with regards to criteria specific to imaging requirements. It was shown that depending on initially set criteria, such as transmitted energy, mechanical index or scanner characteristics, certain pulse combinations offer alternatives to the current imaging modalities and allow to take into account specific constrains due to the targeted application/equipment. Furthermore, the proposed approach is directly applicable in a strict non linear imaging approach, without Doppler processing. An in vitro phantom was designed in order to assess pulse subtraction Doppler for the detection and discrimination of static nonlinear microbubbles in the presence of free flowing ones. It was shown that pulse subtraction Doppler enables such discrimination and the practicability for in vivo situations is discussed. The pulse subtraction Doppler sequences were also tested on a phantom containing magnetic bubbles. It was shown that the magnetic bubbles can be immobilised through a magnetic field to a specific region of interest under flow conditions. The bubbles also showed to be acoustically detectable and to scatter linearly at diagnostic driving pressures. Preliminary work regarding experimental biotinylated microbubbles and their attachment to streptavidin coated surfaces is also presented. Due to their proximity to a wall, researchers have found that targeted microbubbles exhibit different acoustic signatures compared to free ones and this knowledge can improve their detection techniques. The behaviour of microbubbles against a membrane of varying stiffness was also studied through high speed camera observations. It was found both experimentally and by comparison to theoretical modelling that within the stiffness range of human blood vessels the change in acoustical behaviour of microbubbles is negligible. This thesis has taken two complementary research approaches which have shown to constitute advancements for the detection and discrimination of targeted microbubbles

HIGH INTENSITY FOCUSED ULTRASOUND AND OXYGEN LOAD NANOBUBBLES: TWO DIFFERENT APPROCHES FOR CANCER TREATMENT

The study of applications based on the use of ultrasound in medicine and biology for therapeutic purposes is under strong development at international level and joins the notoriously well-established and widespread use of diagnostic applications [1]. In the past few years, High Intensity Focused Ultrasound (HIFU) has developed from a scientific curiosity to an accepted therapeutic modality. HIFU is a non invasive technique for the treatment of various types of cancer, as well as non-malignant pathologies, by inducing localized hyperthermia that causes necrosis of the tissue. Beside HIFU technology, other innovative therapeutic modalities to treat cancer are emerging. Among them, an extremely innovative technique is represented by oxygen loaded nanobubbles (OLNs): gas cavities confined by an appropriately functionalized coating. This is an oxygenating drugs aimed at re-oxygenation of cancerous tissue. Oxygen deficiency, in fact, is the main hallmark of cancerous solid tumors and a major factor limiting the effectiveness of radiotherapy. In this work, these two approaches to treat tumours are under study from a metrological point of view. In particular, a complete characterization of an HIFU fields regarding power, pressure and temperature is provided while oxygen load nanobubbles are synthesized, characterized and applied in in vitro and in vivo experiments

Quantitative characterization of viscoelastic behavior in tissue-mimicking phantoms and ex vivo animal tissues.

Viscoelasticity of soft tissue is often related to pathology, and therefore, has become an important diagnostic indicator in the clinical assessment of suspect tissue. Surgeons, particularly within head and neck subsites, typically use palpation techniques for intra-operative tumor detection. This detection method, however, is highly subjective and often fails to detect small or deep abnormalities. Vibroacoustography (VA) and similar methods have previously been used to distinguish tissue with high-contrast, but a firm understanding of the main contrast mechanism has yet to be verified. The contributions of tissue mechanical properties in VA images have been difficult to verify given the limited literature on viscoelastic properties of various normal and diseased tissue. This paper aims to investigate viscoelasticity theory and present a detailed description of viscoelastic experimental results obtained in tissue-mimicking phantoms (TMPs) and ex vivo tissues to verify the main contrast mechanism in VA and similar imaging modalities. A spherical-tip micro-indentation technique was employed with the Hertzian model to acquire absolute, quantitative, point measurements of the elastic modulus (E), long term shear modulus (η), and time constant (τ) in homogeneous TMPs and ex vivo tissue in rat liver and porcine liver and gallbladder. Viscoelastic differences observed between porcine liver and gallbladder tissue suggest that imaging modalities which utilize the mechanical properties of tissue as a primary contrast mechanism can potentially be used to quantitatively differentiate between proximate organs in a clinical setting. These results may facilitate more accurate tissue modeling and add information not currently available to the field of systems characterization and biomedical research