Adhesion patterns in the microvasculature are dependent on bifurcation angle.

Particle adhesion in vivo is highly dependent on the microvascular environment comprising of unique anatomical, geometrical, physiological fluid flow conditions and cell-particle and cell-cell interactions. Hence, proper design of vascular-targeted drug carriers that efficiently deliver therapeutics to the targeted cells or tissue at effective concentrations must account for these complex conditions observed in vivo. In this study, we build upon our previous results with the goal of characterizing the effects of bifurcations and their corresponding angle on adhesion of functionalized particles and neutrophils to activated endothelium. Our hypothesis is that adhesion is significantly affected by the type of biochemical interactions between particles and vessel wall as well as the presence of bifurcations and their corresponding angle. Here, we investigate adhesion of functionalized particles (2 µm and 7 µm microparticles) to protein coated channels as well as adhesion of human neutrophils to human endothelial cells under various physiological flow conditions in microfluidic bifurcating channels comprising of different contained angles (30°, 60°, 90°, or 120°). Our findings indicate that both functionalized particle and neutrophil adhesion propensity increase with a larger bifurcation angle. Moreover, the difference in the adhesion patterns of neutrophils and rigid, similar sized (7 µm) particles is more apparent in the junction regions with a larger contained angle. By selecting the right particle size range, enhanced targeted binding of vascular drug carriers can be achieved along with a higher efficacy at optimal drug dosage. Hence, vascular drug particle design needs to be tailored to account for higher binding propensity at larger bifurcation angles.

Adhesive interaction of functionalized particles and endothelium in idealized microvascular networks.

OBJECTIVE: Leukocytes play a key role in the early response to tissue injury/infection resulting from physical, chemical or biological stimuli. This process involves the initiation of the leukocyte adhesion cascade mediated by a series of interactions between receptors and ligands on the endothelium and the leukocytes. Here, we characterize the adhesion profile of functionalized particles under physiological flow conditions in an idealized synthetic microvascular network (SMN) characterized by a bifurcation. We hypothesize that differences in the level of adhesion of functionalized particles in bifurcating SMNs are dependent on the ratio of adhesion molecules on the particles as well as geometric features of the in vitro networks. METHODS: Functionalized particles were prepared by coating their surfaces with different ratios of antibodies against ICAM-1 and E-selectin (aICAM-1:aE-selectin=100:0, 70:30, 50:50, 30:70, and 0:100). The adhesion of functionalized particles to 4h TNF-α activated human umbilical vein endothelial cells under shear flow (0.5, 2, and 4dyn/cm(2)) in bifurcating SMNs and in a parallel plate flow chamber was then quantified. RESULTS: The level of adhesion of 50:50 aICAM-1:aE-selectin particles was significantly higher compared to other particles in the bifurcating SMNs (~1.5-4 fold higher). However, in the parallel plate flow chamber 70:30 aICAM-1:aE-selectin particles exhibited a significantly higher level of adhesion (~1.5-2.5 fold higher). Furthermore, the adhesion of particles in junction regions was about 3-18 fold higher than that in straight sections of the SMNs. As expected, in straight sections of the SMNs and in the parallel plate flow chamber particle adhesion increased with decreasing shear. However, particle adhesion did not change significantly with decreasing shear at the junction regions of SMNs for all functionalized particles. CONCLUSION: Adhesion efficiency of functionalized particles is significantly affected by cell-adhesion molecule ratio density as well as geometric features of the vessels. Moreover, the differential adhesion patterns of particles between straight sections of bifurcating SMNs and parallel plate flow chamber, as well as straight sections and junction regions of bifurcating SMNs, indicates that adhesion profile of particles is highly dependent on the vascular geometry of the system used.

A physiologically realistic in vitro model of microvascular networks.

Existing microfluidic devices, e.g. parallel plate flow chambers, do not accurately depict the geometry of microvascular networks in vivo. We have developed a synthetic microvascular network (SMN) on a polydimethalsiloxane (PDMS) chip that can serve as an in vitro model of the bifurcations, tortuosities, and cross-sectional changes found in microvascular networks in vivo. Microvascular networks from a cremaster muscle were mapped using a modified Geographical Information System, and then used to manufacture the SMNs on a PDMS chip. The networks were cultured with bovine aortic endothelial cells (BAEC), which reached confluency 3-4 days after seeding. Propidium iodide staining indicated viable and healthy cells showing normal behavior in these networks. Anti-ICAM-1 conjugated 2-mum microspheres adhered to BAEC cells activated with TNF-alpha in significantly larger numbers compared to control IgG conjugated microspheres. This preferential adhesion suggests that cultured cells retain an intact cytokine response in the SMN. This microfluidic system can provide novel insight into characterization of drug delivery particles and dynamic flow conditions in microvascular networks.

Synthetic tumor networks for screening drug delivery systems.

Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. A key factor is tumor microvasculature with complex effects including convective transport, high interstitial pressure and enhanced vascular permeability due to the presence of "leaky vessels". Current in vitro models of the tumor microenvironment for evaluating drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. In this study, we report on the development of a novel microfluidic platform that models the tumor microenvironment more accurately, with physiologically and morphologically realistic microvasculature including endothelial cell lined leaky capillary vessels along with 3D solid tumors. Endothelial cells and 3D spheroids of cervical tumor cells were co-cultured in the networks. Drug vehicle screening was demonstrated using GFP gene delivery by different formulations of nanopolymers. The synthetic tumor network was successful in predicting in vivo delivery efficiencies of the drug vehicles. The developed assay will have critical applications both in basic research, where it can be used to develop next generation delivery vehicles, and in drug discovery where it can be used to study drug transport and delivery efficacy in realistic tumor microenvironment, thereby enabling drug compound and/or delivery vehicle screening.

A microfluidic impedance flow cytometer for identification of differentiation state of stem cells.

This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells.

Synthetic microvascular networks for quantitative analysis of particle adhesion.

We have developed a methodology to study particle adhesion in the microvascular environment using microfluidic, image-derived microvascular networks on a chip accompanied by Computational Fluid Dynamics (CFD) analysis of fluid flow and particle adhesion. Microfluidic networks, obtained from digitization of in vivo microvascular topology were prototyped using soft-lithography techniques to obtain semicircular cross sectional microvascular networks in polydimethylsiloxane (PDMS). Dye perfusion studies indicated the presence of well-perfused as well as stagnant regions in a given network. Furthermore, microparticle adhesion to antibody coated networks was found to be spatially non-uniform as well. These findings were broadly corroborated in the CFD analyses. Detailed information on shear rates and particle fluxes in the entire network, obtained from the CFD models, were used to show global adhesion trends to be qualitatively consistent with current knowledge obtained using flow chambers. However, in comparison with a flow chamber, this method represents and incorporates elements of size and complex morphology of the microvasculature. Particle adhesion was found to be significantly localized near the bifurcations in comparison with the straight sections over the entire network, an effect not observable with flow chambers. In addition, the microvascular network chips are resource effective by providing data on particle adhesion over physiologically relevant shear range from even a single experiment. The microfluidic microvascular networks developed in this study can be readily used to gain fundamental insights into the processes leading to particle adhesion in the microvasculature.