Alginate modification with the coupling of primary amines relevant for nerve regeneration

Abstract

Alginate is a polysaccharide produced by brown seaweed and some bacteria which is highly demanded in biomedical researches. It can form a gel in presence of divalent cations which has many applications in cell based therapy. Alginate can be used as immobilization matrix for transplanted cells in vivo to protect the transplanted cells against host immune system. It can be modified by coupling with adhesive peptides such as RGD to improve adhesion property for cell entrapment, relevant for tissue engineering including nerve regeneration.Carbodiimide chemistry has been the most general chemical reaction applied to couple functional peptides to alginate. In this study, primary amines were coupled to alginate using both carbodiimide chemistry and a newly developed method of reductive amination and results were analyzed by 1H-NMR. The native stipe alginate was coupled with MeOTyr using carbodiimide chemistry and no detectable coupling was found. But, using reductive amination for coupling MeOTyr to 8% oxidized leaf alginate gave at least 6.5% coupling. Also, the influence of methanol concentration (1%, 10% and 13%) on coupling reaction was assessed and results declared that the coupling degree was not affected by these changes in methanol concentration.Coupling five different types of periodate oxidized alginates (stipe and leaf alginate, mannuronan, poly M and AlgE6/AlgE4 epimerized mannuronan) with MeOTyr model molecule, confirmed that MeOTyr was able to be coupled in highest degree (8.8%) with oxidized mannuronan amongst other types of oxidized alginates. No relationship of the degree of coupling with the content of guluronic acid in the alginate was found.Moreover, GRGDYP, GRGDSP (RGD peptides) and KHIFSDDSSE sequences (a neuropeptid) were coupled to 8% oxidized leaf alginate using reductive amination and gave 4.0%, 4.1% and 5.1% coupling respectively. The large batch of oxidized leaf alginate (46% G) coupled with GRGDSP by using reductive amination was produced for further studies relevant to NSCs encapsulation using this modified alginate.Since the leaf alginate modified in this study might be used for encapsulation of NSCs and endothelial cells in future study, as preliminary study, NSCs and endothelial cells were co-cultured on two different substrates and in two different media. To investigate their co-culture, fluorescence microscopy was used and the live/dead assay confirmed their viability during the co-culture.