Peptide Cross-linked and Grafted Alginates made by Periodate Oxidation, Reductive Amination and Epimerization
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A challenging task in tissue engineering is to imitate the extracellular matrix (ECM) and ensure three-dimensional cellular growth and network formation in vitro. Alginates are linear polymers consisting of (1→4) linked β-D-mannuronate (M) and α-L-guluronate (G), in varying abundance and sequences. Alginate hydrogels cross-linked by divalent ions under physiological terms and with opportunity for chemical modification of properties is a desired biomaterial for tissue engineering. Alginates can be chemically modified e.g. by grafting of peptides that promote cell-adhesion and/or cross-link the chains. Cross-linking the alginate chains with purposive designed peptides can result in a matrix that can be altered by enzymes excreted by the cells that can cleave peptide bonds. This will enable the cells to freely create networks between them, as is done naturally in the body.Here, periodate oxidized mannuronate was cross-linked by a protein cleavable peptide and grafted by cell-adhesion peptides by two different strategies. The two strategies varied only in order of chemical reactions. In the first strategy, G residues was introduced after periodate oxidation of mannuronate and the epimerized POM was thereafter cross-linked and finally grafted with a cell-adhesion promoting peptide (RGD). In the second strategy the POM was first cross-linked and secondly grafted with a cell-adhesion promoting peptide. Finally, G residues were introduced between the oxidized residues by epimerization. To graft peptides to mannuronate/alginate, it was first periodate oxidized to two different degrees, 4% and 8% - implicating that 4 and 8 of 100 residues was oxidized, respectively. Periodate oxidation led to breaks in the M residues and the introduction of two aldehyde functional groups per residue. The peptides were attached to aldehyde groups by reductive amination using 2-Picoline Borane as reducing agent. The cross-linking peptide used contained an amino group at both ends of the peptide a terminal α-amino group and an ε-amino group from the end amino acid lysine (K) which enabled it to be grafted to the alginate at both ends by reductive amination. 1H NMR and UV spectroscopy analyses were done to determine the degree of substitution for all peptides. A fluorescamine assay was done to determine amount of one-end substituted cross-linking peptide. SEC-MALS gave molecular weight and intrinsic viscosity, compression tests gave Young s modulus, syneresis and deformation at fracture of some of the synthesized materials. Cross-linking peptides and cell-adhesion peptides were found to be grafted to all samples. The samples with a degree of oxidation of 8% had the highest degree of substitution and cross-linking. The samples synthesized by strategy 1 had lower average molecular weights compared to the samples synthesized by strategy 2, and also had lower intrinsic viscosities, as a consequence of depolymerization. The cross-linking of mannuronate/alginate chains was confirmed by observation of an increase in molar mass at constant intrinsic viscosities from the non-cross-linked to the cross-linked samples. Compression tests revealed a weakening of strategy 1 sample gels compared to a high G reference gel when mixing the samples with the reference gel (one part sample and three parts high G alginate), thought to be due to low average molecular weight of the samples.Cross-linking of and peptide grafting to mannuronate/alginate was achieved using periodate oxidation and reductive amination. Further work will involve further characterization, up-scaling and studies of cell migration and network formation in the synthesized materials.