Biomimetic sulfated glycosaminoglycans maintain differentiation markers of breast epithelial cells and preferentially inhibit proliferation of cancer cells.

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4-2 (T4-2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue and bromodeoxyuridine proliferation (BrdU) assays, immunofluorescence staining and transwell invasion assay.  Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4-2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In the ELISA assay, a trend of EGF binding was detected for sulfated polysaccharides while QCM-D analysis showed negligible binding of insulin and EGF to sulfated substrates. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells may in part be growth factor dependent. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

Alginate Sulfate Substrates Control Growth Factor Binding and Growth of Primary Neurons: Toward Engineered 3D Neural Networks.

Sulfated glycosaminoglycans (sGAGs) are vital molecules of the extracellular matrix (ECM) of the nervous system known to regulate proliferation, migration, and differentiation of neurons mainly through binding relevant growth factors. Alginate sulfate (AlgSulf) mimics sGAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, thin films of biotinylated AlgSulf (b-AlgSulfn ) are engineered with sulfation degrees (DS = 0.0 and 2.7) and the effect of polysaccharide concentration on FGF-2 and nerve growth factor (ß-NGF) binding and subsequent primary neural viability and neurite outgrowth is assessed. An increase in b-AlgSulfn concentration results in higher FGF-2 and ß-NGF binding as demonstrated by greater frequency and dissipation shifts measured with quartz crystal microbalance with dissipation monitoring (QCM-D). Primary neurons seeded on the 2D b-AlgSulfn films maintain high viability comparable to positive controls grown on poly-d-lysine. Neurons grown in 3D AlgSulf hydrogels (DS = 0.8) exhibit a significantly higher viability, neurite numbers and mean branch length compared to neurons grown in nonsulfated controls. Finally, a first step is made toward constructing 3D neuronal networks by controllably patterning neurons encapsulated in AlgSulf into an alginate carrier. The substrates and neural networks developed in the current study can be used in basic and applied neural applications.

The sulfation of biomimetic glycosaminoglycan substrates controls binding of growth factors and subsequent neural and glial cell growth.

Sulfated glycosaminoglycans (GAGs) are key structural and functional extracellular matrix (ECM) molecules involved in numerous signaling pathways mainly through their interaction with growth factors. Alginate sulfate mimics sulfated GAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, natural biomimetic substrates were engineered by immobilizing biotinylated alginate sulfates with varying degrees of sulfation (DS, from 0 to 2.7) on gold and polystyrene substrates using biotin-streptavidin binding. The build-up of films and the effect of the DS and biotinylation method on FGF-2 binding were assessed using quartz crystal microbalance with dissipation monitoring (QCM-D) and immunohistochemistry. The role of substrate sulfation and FGF-2 loading on the growth of A172 (human glioblastoma multiforme), SH-SY5Y (human neuroblastoma), and PC-12 (rat pheochromocytoma) cell lines was evaluated in vitro using proliferation and neurite outgrowth assessment. An increase in the DS of alginates resulted in augmented FGF-2 binding as evidenced by higher frequency and dissipation shifts measured with QCM-D and confirmed with immunostaining. All sulfated alginate substrates supported the attachment and growth of neural/glial cell lines better than controls with the highest increase in cell proliferation observed for the highest DS (p < 0.05 for all the cell lines). Moreover, FGF-2 loaded substrates with the highest DS induced the most significant increase in neurite-positive PC-12 cells and average neurite length. The developed biomimetic coatings can be used to functionalize substrates for biosensing applications (e.g. gold substrates) and to induce defined cellular responses via controlled growth factor delivery for basic and applied sciences.