(625d) Nanoparticle Uptake in Flow-Conditioned Endothelial Cells Depends  on Glycocalyx Structure and Nanoparticle Shape and Charge

The development of atherosclerosis in arterial blood vessels preferentially occurs at branched, constricted and curved arteries and spans decades. The condition is difficult to treat due to its location and slow progression. The glycocalyx is a sugar-rich extracellular matrix, composed of glycosaminoglycans such as heparan sulfate and sialic acids, with pore sizes of 7 nm or less. These components line the endothelial cells of blood vessels and are critical in maintaining vascular health. The glycocalyx regulates mechanotransduction, chemokine and signaling molecule interactions, and cell-cell communication (Ebong, 2011). In atherosclerotic patients this glycocalyx layer is found to be shed, which suggests that glycocalyx may be linked to cardiovascular disease. Our interest is to leverage unhealthy glycocalyx and its characteristics, combined with nanoscale particle drug delivery vehicles, to develop a preventative approach to atherosclerosis development. To achieve our goal we looked into how the surface and interface properties of the glycocalyx differs under various conditions, and how each component of the matrix affects nanoparticle uptake by blood vessel endothelial cells. A monolayer of human endothelial cells were exposed to shear stress at 15 dynes/cm² for six hours. For a healthy glycocalyx, the cells were cultured with serum-rich media, while a collapsed glycocalyx was modeled through serum-deficient media. Glycocalyx cleaving enzymes were perfused across the endothelial cells for degraded models (Fels, 2014). Heparan sulfate was degraded using heparinase III enzyme at 45 mU/mL (U is sigma units) and sialic acid residues were removed through neuraminidase at 1215 mU/mL. After degradation, the shed components were introduced back into the culture at concentrations found in a healthy glycocalyx to simulate repairing of the matrix. After exposure to flow and glycocalyx treatments, the endothelial cells were incubated for 16 hours with various gold nanoparticles coated in polyethylene glycol and AlexaFluor 647 fluorophore, then imaged through confocal microscopy to determine glycocalyx structure and nanoparticle uptake. The particles used were gold nanospheres with diameters of 10 nm and nanorods with diameters of around 20 nm, with charges ranging from -10 mV to -40 mV. These characteristics allow for the particles to localize at dysfunctional glycocalyx - a healthy glycocalyx would discourage uptake of particles that are negatively charged and/or larger than 7 nm because of the pore size cutoff and innate negative charge of the matrix. The glycocalyx of the human endothelial cells were found to be more prominent and continuous with flow conditioning, as imaged by bovine serum albumin, heparan sulfate, and sialic acid immunostaining. This matches results by Dewey et al. with regards to heparan sulfate regrowth under laminar shear flow. The thickness and coverage of all stains increased after pre-treatment with flow stimulus and coincided with minimal nanoparticle uptake as expected. However, after serum deprivation or enzyme treatments the uptake of gold nanoparticles increased significantly because of glycocalyx disruption. Addition of the degraded components back into the culture during the nanoparticle treatment decreased the uptake. Further, localization of the nanoparticles were observed to be different within the monolayers depending on how the glycocalyx was degraded. These results suggest an importance in consideration of the glycocalyx layer in observing nanoparticle interaction and uptake for vascular drug delivery. The charge and size of the particles have been previously identified as important factors in uptake kinetics. However, here we show the proper experimental modeling of the cellular conditions and glycocalyx in the form of flow stimulus and degradation is also necessary to accurately predict how the cells interact with nanoscale particles. Also, the ability to target compromised glycocalyx that is prone to atherogenesis is an important first step in reducing plaque development and cardiovascular disease prevention.