Development of a glycan-based platform for the elucidation cell-glycosaminoglycans interactions

The chemical and structural diversity, molecular weight (MW) and patterns of glycosaminoglycans (GAGs) in the extracellular matrix (ECM) are important factors in cell's behaviour [1,2]. In the ECM, GAGs are commonly presented as conjugates with proteins or lipids, which are prone to interact with cells, playing different bioroles, including modulators cell-ECM and cell-cell interactions. The binding of GAGs to their receptors is driven by weak and often multivalent interactions. Generally, these interactions are hard to detect and measure [3, 3]. State of art studies involved the use ofGAGs in solution, associated with higher freedom of mobility as compared with GAGs immobilized in ECM. Heerein, we propose an alternative design based on end-on immobilized GAGs. The developed platform is versatile, as it allows immobilization of different GAGs, and offers possibility to study their interactions with cells in a high-throughput fashion using complementary label-free techniques. Continuous gradients were assembled on APTES silanized glass using gold nanoparticles (20 nm). GAGs, functionalized with alkanothiol (C₁₁SH) at the reducing end were covalently bound to the goldgradients as confirmed by water contact angle, x-ray spectroscopy (XPS), and fluorescence microscopy. The stability and biofunctionality of the immobilized GAGs was confirmed by studing their interactions with lectin - wheat germ agglutinin that is specific towards N-acetyl-D-glucosamine, was used in this studies. Perliminary evaluation of the interactions of hyaluronan platforms with 3 breast cancer cell lines that differ by their expression of CD44 were also perfomed. The tested cells recognize the gradient and more attached cells were observed at higher densities of hyaluronan. Cell morphology was also affected - cell typically spread on areas richer in hyaluronic acid. Of note, these differences were more pronounced for cells with lower expression of CD44, thus, allowing further application of the developed platforms for cell separation.