SEC determination of cross-link efficiency in hyaluronan fillers

The cross-link efficiency in hyaluronan fillers was determined by means of triple detector (RI, LALS-RALS and a Differential Viscometer) GPC/SEC in aqueous buffer. The low water solubility of HA cross-linked with BDDE (HBC) was overcome with an alkaline hydrolysis step. The kinetics of linear HA hydrolysis was investigated at different NaOH concentrations (0.25–0.5–1 M), initial polymer Mws (200–700–1000–1200–1600 kDa) and polymer concentrations (0.1–0.3–0.5 mg/ml). As expected for first-order kinetics, the apparent hydrolysis constant (kh) was independent of polymer concentration and initial Mws. The kh was found to be linearly dependent on the NaOH concentration, suggesting a random polymer degradation. A similar behavior was observed for HBC polymers, synthesized with 5–7.5–10–14–18% mol/mol of BDDE for HA repeat unit. The degree of crosslinking was obtained using the Zimm–Stockmayer equation for random, tri-functional polydisperse polymers. The contraction factor (g) was determined after an accurate experimental measurement of the structure factor (ɛ). Comparative studies were performed by 1H NMR spectroscopy and rheological measurements. Interestingly, the number of effective cross-links found is very low compared to the total BDDE linked; i.e. in a HBC sample only 0.04% of total BDDE linked (equal to 4.6% by 1H NMR analysis) gives an effective cross-link.