Hyaluronan size and concentration: Effect on key biophysical and biochemical features.

The effect of hyaluronan (HA) molecular weight (MW) and concentration (c) on key features of HA-formulations was systematically studied, in vitro, exploring the widest range/number of MW/c to date. Nine pharmaceutical grade HA samples (60-2500 kDa) were hydrodynamically characterized using Size Exclusion Chromatography-Triple Detector Array (SEC-TDA) also providing conformational data. HAs aqueous solutions (thirteen concentrations in the range 0.1-32 g/L) were tested for dynamic viscosity (η). η dependence on MW/c was analyzed providing mathematical correlations not only for the specific zero-shear viscosity, but also for the critical shear rate and the shear-thinning-extent. Besides confirming the dilute and semi-dilute c-regimes for the HAs, a third concentrated regime was evidenced for the 220-2500 kDa samples. Data on how MW affects the dependence of viscosity parameters on c and vice-versa were provided. The 60-90 kDa HAs proved stable to thermal sterilization and enzymatically catalyzed hydrolysis, while the 220-2500 kDa samples depolymerized to an extent depending, beyond concentration, on MW. HA size did not significantly affect fibroblasts behavior: under the conditions here tested, the HAs similarly sustained human dermal fibroblasts growth and wound healing also showing comparable effect on collagen-I, elastin and hyaluronan synthase-1 expression. Overall, results valuably contribute to the understanding of the HA MW/c impact on the rheological, stability and biochemical features of the final formulations, also providing mathematical correlations allowing for their optimization towards specific performance.