The chemical and structural diversity and glycosaminoglycans (GAGs) profile in the extracellular matrix (ECM) are important factors in cells' stemness, differentiation, development, and thus, in the advancement and severity of different diseases [1, 2]. In the ECM, GAGs are commonly presented as conjugates with proteins or lipids, which are prone to interact with transmembrane proteins having different bioroles, including as modulators cell-ECM and cell-cell interactions. The binding of GAGs to their receptors is driven by weak and often multivalent interactions, therefore hard to detect and measure [3, 4]. State of the art studies involved the use of GAGs in solution, with higher freedom of mobility as compared with the native GAGs immobilized in ECM.
Herein, we propose an alternative design that is based on end-on immobilized GAGs in a high-throughput platform that offers a possibility to study the interactions of different GAGs with cells using complementary label-free techniques. Continuous colloidal gradients were assembled on silanized glass using gold nanoparticles (20 nm). GAGs functionalized with alkanthiol (C11SH) at the reducing end were covalently bound to the gold gradients as confirmed by water contact angle, x-ray spectroscopy and fluorescence microscopy. The stability and biofunctionality of the immobilized GAGs was confirmed by studying their interactions with lectin - Wheat germ agglutinin that is specific towards N-acetyl-D-glucosamine was used in these studies. Preliminary evaluation of the interactions of hyaluronan platforms with 3 breast cancer cell lines that differ by their expression of CD44 were also performed. The tested cells recognize the gradient and more attached cells were observed at higher densities of hyaluronan. Cell morphology was also affected - cells 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.
Acknowledgements: The authors thank the Portuguese FCT (Grants no: SFRH/BD/114847/2016, SFRH/BD/114847/2016, IF/00032/2013 and IF/00373/2014), EU (ComplexiTE ERC-2012-ADG 20120216-321266, H2020-TWIN-CHEM2NATURE-692333 and EuroNanoMed CytoNanoHeal) for financial support.
References:
[1] D. Soares da Costa, R. L. Reis, and I. Pashkuleva, Annual Review of Biomedical Engineering, 19, 1 (2017)
[2] H.E. Bulow and O. Hobert Annual Review of Cell and Developmental Biology, 22, 1 (2006)
[3] P.R. Crocker and T. Feizi, Current Opinion in Structural Biology, 6, 5 (1996)
[4] J.C. Paulson, O. Blixt and B.E. Collins, Nature Chemical Biology, 2, 5 (2006)