Alginate is widely used in the biomedical field, particularly to build three-dimensional (3D) systems with ECM-like properties. Despite being a bio-inert biomaterial, alginate can be chemically modified to promote highly specific cell-ECM interactions. We have shown that molecularly designed alginate 3D matrices recap key features of native ECMs supporting tissue morphogenesis[2-4]. Here, we developed a novel methodology to synthesize and characterize functional bioresponsive alginate hydrogels based on thiol–maleimide “click” chemistry. The potential of these peptides-conjugated hydrogels as ECM-like matrices for 3D cell culture was evaluated.
Ultra-pure low viscosity alginate (Alg) with high guluronic content was modified with variable amounts of maleimide (AlgM) by amidation of alginate carboxyl groups with the amine groups of 1-(2-Aminoethyl)-maleimide. Degree of substitution was assessed by 1H-NMR. Thiol-flanked (bi-functional) protease-sensitive peptide (GIW-peptide, CGPQGIWGQC) and thiol-terminated cell-adhesion peptide (RGD-peptide, CGGGGRGDSP) were grafted to AlgM via thiol-maleimide Michael addition click reaction. To confirm peptide double-end grafting and consequent crosslinking, Alg solutions dynamic viscosity was analyzed by oscillation/viscometry rheology. Alterations on GIW-crosslinked AlgM viscosity and molecular weight were assessed by gel permeation chromatography (GPC). Grafting of RGD-peptide was quantified by BCA Protein Assay (Pierce). Primary human mammary normal (nFIB) and cancer-associated (CAF) fibroblasts mixed with alginate and peptide solutions were cast as hydrogels. 3D cell response to different GIW/RGD-peptide concentrations was studied at different timepoints. MMP production (gelatin zymography), cell viability (live-dead assay), morphology (F-actin staining), and mechanical properties were assessed.
1H-NMR qualitatively confirmed successful alginate functionalization with different maleimide amounts (theoretical substitution degree from 1 to 10%). Maleimide presence was identified by the appearance of a new peak (~6.9ppm) corresponding to the protons in the double bond of the maleimide group. Reaction efficiency was approximately 10%. We observed that high degrees of maleimides lead to poor solubility of alginate derivatives, so we only used derivatives with up to 0.3% of modification. Addition of bi-functional GIW-peptide increased hydrogel viscosity due to the formation of a chemically crosslinked gel network. Viscosity increase and consequent gel formation was observed between 120-480µM of GIW-peptide. Higher concentrations (840µM) did not alter solution viscosity but increased alginate Mw, because GIW-peptides were preferentially bound only by one side, occupying the maleimides without bridging two alginate chains. nFIB and CAF (fibroblasts with different proliferative/metabolic profiles) were successfully embedded within GIW/RGD-AlgM matrices, presenting elongated morphologies and forming extensive multicellular networks, contrary to control MMP-insensitive hydrogels, where cells remained essentially round. Due to its nature, CAF formed more extensive cellular networks faster, without major differences regarding MMP production.
A novel methodology for the synthesis of alginate containing maleimide functional groups was established. Covalently grafted maleimides allow alginate biofunctionalization and in situ crosslinking by thiol-Michael addition reaction. Incorporating protease-sensitive peptides significantly enhanced 3D cell-cell interactions in alginate hydrogels, improving their performance as ECM-mimics.
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