Optimalised Carbodiimide Chemistry for RGD-coupled Alginate

Alginate is a naturally ocurring polyanion of (1→4)linked β-d-mannuronic acid (M) and its C-5 epimer α-l guluronic acid (G). The polyanion, and particularly long stretches of guluronic acids, chelates and form hydrogels in the presence of divalent cations such as Ca2+ . Chelation occur at physiological conditions and the formed hy- drogels are biocompatible and stable. These properties nominates alginate hydrogels as promising biomaterials in tissue engineering applications. Encapsulation of cells in alginate beads is easily prepared by mixing cells with high molecular weight alginate, and dripping the solution into a CaCl2 solution. When alginate comes into contact with Ca2+ ions, alginate beads are immediately formed and the cells are captured in a three dimensional alginate matrix. Once inside the alginate capsule, the cells can be transplanted into a host deficient in the particular cells. The alginate protects the cells against the immune system of the host, opening for allograf transplantation without immunosuppressiva. The pores in the alginate network are big enough to enable diffusion of waste and nutrition through the membrane, which is important for cell survival. Alginate is in itself not cell adhesive. However, alginate can be tailored into a cell adhesive biomaterial by attachement of cell adhesive peptides, such as the RGD motif found in extracellular matrix molecules such as fibronectin. Coupling of cell adhesive peptides increase cell survival in three dimensional alginate matrices. In this study, RGD-alginate is tailored with a chemoenzymatic approach that ensures RGD-coupling to non-gelling residues. This procedure starts with non-gelling man- nuronan that is chemically modified by attachement of the cell adhesive peptide GRGDYP by carbodiimide chemistry. Gelling residues are introduced to peptide coupled mannuronan by a two step epimerisation catalyzed by the epimerases AlgE4 and AlgE6. In this study, the carbodiimide chemsitry used for coupling GRGDYP to mannuro- nan is optimalised to create alginate with more than 0.2% bound peptide. The model molecules fluoresceinamine, 4-aminophenol and L-tyrosine-methyl ester are used for optimalisation, and the latter model molecule was found to give the best representation of peptide coupling to mannuronan. The carbodiimide mediated cou- pling to mannuronan is investigated by varying the pH, temperature and reactant concentrations. The degrees of coupling for each intervention is assessed by 1 H NMR and UV/vis spectroscopy. The presence of covalently bound by-products, named N-acylurea adducts, to mannuronan is assessed by 1 H NMR spectroscopy and a controlled reduction of these unwanted compounds is attempted. The results from the optimalisation indicates that coupling of Me-O-Tyr and GRGDYP to mannuro- nan is concentration-dependent, as an increase in coupling was observed when the GRGDYP and Me-O-Tyr concentrations was increased. The highest peptide incor- poration described in this study was 3.4% GRGDYP coupled to mannuronan, which is higher compared to similar studies. The optimalised carbodiimide chemistry is applied to a large scale batch of RGD- coupled alginate that is to be used for cell encapsulation of olfactory ensheating cells from neonatal rat brain. High molecular weight alginate was coupled with 0.45% GRGDYP and filtrated with active coal before cell encapsulation. The coal filtration removed a substantial amount of the unwanted N-acylurea adducts but also removed peptides, resulting in a GRGDYP coupling of 0.1% calculated from UV/vis spectroscopy, and 0.4% calculated from 1 H NMR spectroscopy. This indicates that peptides are associated with N-acylurea adducts and that active coal filtration is necessary to remove them. Encapsulation of olfactory ensheating cells to RGD-coupled alginate gave no mor- phology changes or enhanced cell viability compared to non-peptide coupled alginate. It is believed that the low enzymatic action of the AlgE6 epimerase, a low concen- tration of peptides or a combination of both, have influenced the cell viability and lack of morphology changes. Enhanced peptide incorporation can be achieved by increasing the reactant concentrations of the peptides, but this would lead to a more expensive procedure. Increased efficacy, meaning a higher peptide coupling with lower adduct formation, at lower peptide concentrations was not achieved. However, the use of sodium borohydride in combination with periodate oxidised alginates for peptide coupling should be assessed as a novel approach for increased peptide yields at lower peptide concentrations.