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

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

Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 μM and 10.1 μM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 μM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses

Evaluation of the effects of a dynamic culture on osteogenic differentiation of oral-periosteal cells grown on PLGA sponges

Oral-periosteum derived stem cells represent an innovative cell source for bone tissue engineering applications in terms of accessibility and self-commitment towards osteogenic lineage [1]. In this scenario, biomaterials play a pivotal role in tissue engineering in supporting stem cells growth and regeneration of tissue defects [2]. Among these biomaterials, Fisiograft®, a synthetic co-polymer composed of polylactic and polyglycholic acids produced by Ghimas (Bologna, Italy), is highly biocom- patible and completely absorbed within 4-6 months. In particular, Fisiograft® sponges are normally used in dental applications to fix completely periodontal defects without damage Schneider’s membrane. We evaluated the osteogenic potential of Fisiograft® sponges on oral-periosteal cells derived from patients undergoing dental extractions. For this purpose, we created a dynamic culture based on a rotating apparatus in which we seeded periosteal cells with Fisiograft® sponges for 7, 14 and 21 days without adding osteogenic supplement in the medium. Osteoblast differen- tiation of cells was evaluated by Alizarin Red S staining and by qRT-PCR on genes involved in bone development. Results show that Fisograft® sponges promote greater osteogenic differentiation of cells in the dynamic culture with respect to standard condition already at 14 days, as demonstrated by Alizarin Red staining. BMP-2 and Osteoprotegerin genes are highly expressed by cells grown on Fisiograft® sponges in dynamic culture at 14 days with respect to plastic culture. Taken together, these results confirm the osteogenic potential of Fisiograft® sponges in accelerating the dif- ferentiation of cells to an osteoblast phenotype (already to 14 days of culture) without any osteogenic induction. The combination of this PLGA biomaterial and oral-peri- osteal cells could represent a promising bio-complex in maxillo-facial tissue repair

Organocatalytic vinylogous Mannich reaction of trimethylsiloxyfuran with isatin-derived benzhydryl-ketimines

A family of chiral quaternary 3-aminooxindole butenolides has been synthesized by BINOL-derived phosphoric acid-catalyzed addition of trimethylsiloxyfuran to isatin-derived ketimines. Such a vinylogous Mannich-type reaction was found to produce diastereoisomeric butenolides in good yields and in most cases high enantiomeric excesses. The configurational assignment of the obtained products was safely performed by chemical correlation. A computational study of the transition state allowed rationalizing the obtained stereochemical outcome, highlighting the possible binding modes of the catalyst-imine-nucleophile transition complex

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

One step access to oxindole-based β-lactams through Ugi four-center three-component reaction

A multicomponent Ugi reaction involving isatin, isocyanide and β-amino acid components has been developed