Investigating triazine-based modification of hyaluronan using statistical designs

Hyaluronan (HA) and its derivatives have been extensively researched for many biomedical applications. To precisely tailor the property of HA by derivatizing it to a pre-determined extent is challenging, yet critical. In this paper, we used 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) to derivatize HA via a triazine-based coupling reaction. Using a fractional factorial (FF) design, we observed that water content in the solvent, and molar ratios of CDMT and NaHCO3 to the carboxylate were the significant factors controlling the derivatization. We investigated how the effect of each factor changes as reaction conditions change. Moreover, by altering the amount of CDMT and NaHCO3, we developed a cubic regression model for precise control of the extent of derivatization using a response surface methodology (RSM) with a D-optimal design. No spurious peaks were detected by 1H NMR spectrum and only 10% decrease of molecular weight of the derivatized HA was determined by GPC. The HA with 6% modification was relatively biocompatible up to 15 mg/mL.