Continuous Measurement of Biological Noise in <em>Escherichia Coli</em> Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

<em>In Vitro</em> 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Technion - Israel Institute of Technology

Technion - IIT

In this review we focus on shear targeted drug delivery as a novel strategy to selectively deliver drugs to sites of vascular obstruction. We review the physics of stenotic (abnormally narrowed) blood vessels, while focusing mainly on the hemodynamics and transport phenomena at these sites. We then discuss how the local abnormal levels of shear stress, which can mechanically activate platelets, can be leveraged for localized drug delivery. We describe the development of Shear Activated Nano-particle Aggregates (SA-NPAs) that are designed to release and localize their nanoparticle drug carriers at sites of vascular stenosis. We present results in a variety of in vivo models, demonstrating the superiority of SA-NPAs carrying a thrombolytic drug compared to conventional treatment with the free drug. We also describe, shear-stress sensitive lenticular liposomes, which also show selective release under stenotic flow conditions. We then discuss limitations of both technologies, challenges in this new field and potential future applications. Altogether, we believe that mechano-sensitive therapeutics may offer a potential new approach for treatment of cardiovascular diseases.

Shear targeted drug delivery to stenotic blood vessels.

Intracranial side aneurysms (IA) are pathological blood-filled bulges in cerebral blood vessels. Unlike healthy blood vessels where mass transport is dominated by convection, both diffusion and convection can play an active role in aneurysm sites. Here, we study via dye washout experiments and numerical simulations, the transport characteristics of particles (1 micron) and small molecules (300 Da) into simplified side aneurysms models following bolus injection. Time-lapse fluorescent microscopy imaging performed in our idealized aneurysm models showed that the parent artery geometry (located on the inner vs. outer curvature) as well as the aneurysm aspect ratio (AR) affect the washout kinetics while the pulsatile nature of the flow, maintained within the physiological range, carries only a minor effect. Importantly, in the absence of effective diffusion, particles that are located on slow streamlines linger within the aneurysm cavity, a phenomenon that could be of importance in deposition of cells and nano/micro-particles within aneurysms. Altogether, mass transport studies may provide valuable insights for better understanding of aneurysm pathophysiology as well as for the design of new diagnostic and theranostic nano-medicines.

Mapping the Transport Kinetics of Molecules and Particles in Idealized Intracranial Side Aneurysms.

In arterial thrombosis, von Willebrand factor (VWF) bridges platelets to sites of vascular injury. The adhesive properties of VWF are controlled by its different domains, which may be engineered into ligands for targeting nanoparticles to vascular injuries. Here, we functionalized 200 nm polystyrene nanoparticles with the VWF-A1 domain and studied their spatial adhesion to collagen or collagen-VWF coated, real-sized coronary stenosis models under physiological flow. When VWF-A1 nano-particles (A1-NPs) were perfused through a 75% stenosis model coated with collagen-VWF, the particles preferentially adhered at the post stenotic region relative to the pre-stenosis region while much less adhesion was detected at the stenosis neck (~ 65-fold less). When infused through collagen-coated models or when the A1 coating density of nanoparticles was reduced by 100-fold, the enhanced adhesion at the post-stenotic site was abolished. In a 60% stenosis model, the adhesion of A1-NPs to collagen-VWF-coated models depended on the location examined within the stenosis. Altogether, our results indicate that VWF-A1 NPs exhibit a flow-structure dependent adhesion to VWF and illustrate the important role of studying cardiovascular nano-medicines in settings that closely model the size, geometry, and hemodynamics of pathological environments.

The Flow Dependent Adhesion of von Willebrand Factor (VWF)-A1 Functionalized Nanoparticles in an in Vitro Coronary Stenosis Model.

Targeted drug delivery to diseased vasculature, such as atherosclerotic lesions, is a multistep process, which is based on the transport of drug carriers to a selected region and their deposition at the desired destination. Current modeling approaches, including microfluidics and animal models, fail to accurately simulate this multi-scale process in human arteries, where blood flow is dominant. Here we study particle deposition in endothelialized 3D reconstructed models of the human carotid bifurcation under physiological hemodyamic conditions. Our results showed that particle localization is highly dependent on vessel geometry and local flow features. Additionally, while strongly adhesive particles tend to adhere more profoundly at high-shear regions, associated with athero-thrombosis, enhanced deposition at vascular flow regions, associated with inflammation and plaque accumulation, e.g., recirculation flows, can be achieved using weakly adhesive particles. Moreover, pulsatile flow as well as presence of blood cells significantly reduce particle adhesion and affect their deposition pattern. These findings highlight the key role of vessel geometry, hemodynamics and particle characteristics in the optimizing vascular targeting nano-carriers.

Mapping deposition of particles in reconstructed models of human arteries.

Thrombus formation at athero-thrombotic sites is initiated by the exposure of collagen followed by platelet adhesion mediated by the platelet-specific collagen receptor glycoprotein VI (GPVI). Here, dimeric GPVI was used as a targeting motif to functionalize polymeric nanoparticle-based drug carriers and to show that with proper design, such GPVI-coated nanoparticles (GPNs) can efficiently and specifically target arterial injury sites while withstanding physiological flow. In a microfluidic model, under physiological shear levels (1-40 dyne/cm), 200 nm and 2 μm GPNs exhibited a >60 and >10-fold increase in binding to collagen compared to control particles, respectively. In vitro experiments in an arterial stenosis injury model, subjected to physiological pulsatile flow, showed shear-enhanced adhesion of 200 nm GPNs at the stenosis region which was confirmed in vivo in a mice ligation carotid injury model using intravital microscopy. Altogether, our results illustrate how engineering tools can be harnessed to design nano-carriers that efficiently target cardiovascular disease sites.

Glycoprotein VI (GPVI)-functionalized nanoparticles targeting arterial injury sites under physiological flow.

Intracranial aneurysms frequently develop blood clots, plaque and inflammations, which are linked to enhanced particulate mass deposition. In this work, we propose a computational model for particulate deposition, that accounts for the influence of field forces, such as gravity and electrostatics, which produce an additional flux of particles perpendicular to the fluid motion and towards the wall. This field-mediated flux can significantly enhance particle deposition in low-shear environments, such as in aneurysm cavities. Experimental investigation of particle deposition patterns in  models of side aneurysms, demonstrated the ability of the model to predict enhanced particle adhesion at these sites. Our results showed a significant influence of gravity and electrostatic forces (greater than 10%), indicating that the additional terms presented in our models may be necessary for modelling a wide range of physiological flow conditions and not only for ultra-low shear regions. Spatial differences between the computational model and the experimental results suggested that additional transport and fluidic mechanisms affect the deposition pattern within aneurysms. Taken together, the presented findings may enhance our understanding of pathological deposition processes at cardiovascular disease sites, and facilitate rational design and optimization of cardiovascular particulate drug carriers.

Computational and experimental investigation of particulate matter deposition in cerebral side aneurysms.

From weakly adhesive to highly thrombogenic: the shear gradient switch.

Drug carriers for targeting cardiovascular diseases have been gaining a respectable attention, however, designing such carriers is challenging due to the biophysical complexity of the vascular system. Wall shear stress (WSS), exerted by blood flow on the endothelium surface, is a crucial factor in the circulatory system. WSS affects the adhesion and preferential accumulation of drug carriers. Here, we propose, an innovative approach to investigate particle adhesion in a converging artery-sized model, lined with human endothelial cells. Unlike widely used microfluidic and in vivo setups, our model enables to investigate particle accumulation in a continuous WSS range, performed in a single experiment, and at the right scale relevant for human arteries. First, we characterized the flow and the WSS map along the designed model, which can span along the entire arterial WSS range. We then used the model to examine the effect of particle size and the suspension buffer on particle adhesion distribution. The results demonstrated the role of particle size, where the same particles with a diameter of 2 µm exhibit shear-decreased adhesion while 500 nm particles exhibit shear-enhanced adhesion. Furthermore, under the same WSS, particles show a similar behavior when suspended in a Dextran buffer, having a viscosity analogous to blood, compared to a phosphate buffer solution without Dextran. Moreover, experiments with RBCs in the phosphate buffer, at a 40% physiological hematocrit, decreased particle adhesion and affected their deposition pattern. Altogether, our study suggests an original platform for investigating and optimizing intravascular drug carriers and their targeting properties.

Netanel Korin

Department of Biomedical Engineering

Technion - Israel Institute of Technology

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Netanel Korin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

Technion - Israel Institute of Technology

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Department of Biomedical Engineering