Hyaluronic acid sodium salt from

Hyaluronic acid sodium salt from Streptococcus equi (mol wt 1.5–1.8 MDa, ≤1% protein), adipic acid dihydrazide (AAD), 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC), and cellulose dialysis tubing (12,400 Da) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All other chemicals were supplied by Lach-Ner (Neratovice, Czechia) at reagent grade. Hyaluronic acid sodium salt was dissolved in deionized water (prepared using TKA Smart2Pure, Thermo Fisher Scientific, Waltham, MA, United States) to create a 2.5 mg/ml solution. To prepare 5 mg of HANPs, 2 ml of HA solution was used and afterwards 3.4 ml of acetone was added to the solution (15 min incubation, gentle stirring). After the addition of EDC [2.5 mg/75 μl ultrapure water (prepared by TKA Smart2Pure)] and AAD (2.5 mg/75 μl), acetone was added in a stepwise manner (2.03 ml per step) in three repetitions. The total acetone/water ratio was set to 279/80 (w/w) and surplus acetone was then removed by evaporation at 60°C with following dialysis against 0.9% NaCl solution.