Composite glycopeptide nanofiber-poly(ethylene glycol) hydrogels to probe extracellular - interactions Antonietta Restuccia, Inha Baek, Matthew D. Molinaro, Gregory A. Hudalla J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida

Statement of Purpose: are carbohydrate-binding that modulate cell phenotype and function in various healthy and pathological processes via recognition of extracellular . (ECM) , such as laminin and collagen IV, were identified as lectin-binding partners more than 30 years ago, yet our understanding of the biological role of lectin- ECM interactions remains limited. This is because studies of lectin-ECM interactions primarily rely on naturally- derived reagents having ill-defined and variable glycan content (e.g., Matrigel), or lectin inhibitors that indiscriminately disrupt lectin-ECM and lectin-cell interactions. To address these challenges, herein we describe synthetic biomaterials with user-defined glycan content as a platform to probe lectin-ECM interactions independently of lectin-cell interactions. Specifically, we fabricate composite hydrogels consisting of poly(ethylene glycol) diacrylate (PEGDA) and our established lectin- binding nanofibers based on self-assembling Figure 1. (a) Schematic of PEGDA and PEGDA-GQ11 carbohydrate-modified peptides (i.e., “glycopeptides”) hydrogels. (b) Binding and (c) release of WGA from [1]. A key feature of our glycopeptide nanofibers is that PEGDA and PEGDA-nanofiber hydrogels. (d) Binding of their lectin-binding specificity and affinity can be tailored GFP to PEGDA and PEGDA-nanofiber hydrogels. by varying the type and density of pendant carbohydrate. with 1 mM non-glycosylated peptide nanofibers (NQ11) Methods: A glycosylated variant of the self-assembling or hydrogels lacking nanofibers (Fig. 1b). The amount of peptide QQKFQFQFEQQ (Q11) terminated with the WGA absorbed by hydrogels with glycopeptide monosaccharide n-acetylglucosamine (i.e., “GQ11) was nanofibers scaled with GlcNAc content and could be synthesized using standard solid-phase protocols. predicted from WGA:GlcNAc binding affinity. In Nanofibers were prepared by combining GQ11 and non- contrast, WGA absorbed by hydrogels with NQ11 glycosylated Q11 in water at different molar ratios. N- nanofibers or no nanofibers matched that expected at acetylglucosamine on GQ11 was converted to n- equilibrium due to passive diffusion (Fig. 1b dashed line). GalNAc-beta-1,4-GlcNAc (LacDiNAcQ11) via a GalNAc PEGDA-GQ11 hydrogels released predictable microgram transferase and UDP-GalNAc donor. Hydrogels were quantities of WGA over multiple days, whereas WGA fabricated by combining 150 mg/mL PEGDA (8 kDa) was completely released from PEGDA within hours (Fig. with 1 mM glycopeptide nanofibers and 0.05% Irgacure 1c). Notably, PEGDA-GQ11 hydrogels and PEGDA 2959, spotting this solution onto hydrophobicized glass hydrogels absorbed a similar mass of green fluorescent slides, and then exposing to UV for 10 min. Hydrogels (GFP) (Fig 1d), demonstrating that WGA binding were removed from the glass slides and placed into a bath to PEGDA-GQ11 hydrogels was specific. Likewise, of 1x phosphate-buffered saline (PBS) containing lectin galectin-3 was selectively absorbed and released by (e.g. , galectin-1, or galectin-3). PEGDA-LacDiNAcQ11 nanofibers (not shown). After 24 h incubation, lectin-containing buffer was removed and analyzed for lectin content as an indirect Conclusions: PEGDA hydrogels fabricated in the measure of hydrogel loading efficiency. Hydrogels were presence of glycopeptide nanofibers selectively capture then placed in excess fresh 1x PBS, and lectin content of and retain soluble lectins analogous to natural the overlaid buffer was analyzed at various time points extracellular matrices with integrated components. Unlike conventional research tools to study Results: PEGDA hydrogels formed in the presence or lectin-ECM interactions, the hydrogels reported here absence of glycopeptide nanofibers had similar provide user-defined glycan content, which enables fine morphology, physical properties, and optical properties. control of the type and concentration of lectin that can be To characterize lectin binding properties of composite bound, as well as the rate at which the lectin is released. PEGDA-glycopeptide nanofiber hydrogels, we first We envision that these materials will provide useful tools characterized the absorption and release of wheat germ to study the role of lectin-ECM interactions on lectin agglutinin (WGA), a GlcNAc-binding plant lectin (Fig 1). regulation of cell phenotype and function.

Hydrogels with 1 mM GQ11 nanofibers absorbed [1] Restuccia, A., Tian, Y.F., Collier, J.H., Hudalla, G.A. significantly more WGA at equilibrium than hydrogels Cellular and Molecular Bioengineering 8, 471 (2015).