Dialysis membrane tube

A total of 264 mg of sodium hyaluronate (0.66 mmol of carboxylate group) was dissolved in deionized water (52 mL) before the addition of acetonitrile (35 mL). The homogeneous solution was cooled for 0.5 h in an ice bath, and then 347.5 mg of CDMT (1.98 mmol) was added. One hour later, 258.5 mg of Arg-OMe^. 2 HCl (0.99 mmol) were added. After adjusting the pH value to 6.5 with 0.5 M NaOH, 217.8 μL of NMM (1.98 mmol) was added, and the reaction was then left under constant stirring (about 400 rpm) overnight. The HA–Arg product was then purified against H₂O for 24 h, followed by NaCl (0.1 M) for 2 days, and finally against water for 2 days, using a dialysis membrane tube with a Mw cut-off of 3.5 KDa (Thermo Fisher Scientific, Rockford, Illinois, USA); finally, the product was lyophilized using a Lio-5P lyophilizer (Vetrotecnica Srl, Padua, Italy). The final product was obtained as a white spongy material with a yield of 76% (290 mg), and it was kept under refrigeration until use. Note: ¹H NMR (400 MHz, D₂O) δ ppm: 4.40 (m, 3H, O–CH–O;—CH α), 3.95–3.10 (m, 15H, C–CH–O; –OCH₃), 3.107 (m, 2H, CH₂δ), 1.97 (s, 3H, CO–CH₃–N), 1.80–1.43 (m, 4H, C–CH₃–CO and –CH₂).

Hyaluronate (HA) was purchased from Lifecore Co. (Chaska, MN). Botulinum toxins (BTX, Meditox, Ochang, Korea) were kindly gifted from Dr. Jeesoo An at the Wellman Center for Photomedicine in Massachusetts General Hospital. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was obtained from Tokyo Chemical Industry (Tokyo, Japan). N-hydroxysulfosuccinimide (sulfo-NHS), phosphate buffered saline (PBS), diaminohexane (DAH) and hydroxylbenzotriazole (HOBt) were purchased from Sigma (St. Louis, MO). Dialysis membrane tube was obtained from Thermo Scientific Co. (Waltham, MA).

Chitosan (0.3 g, 1.9 mmol of an average monomer unit) was completely dissolved in 2 wt% aqueous acetic acid (7.5 mL), followed by the addition of OS (30 mg) dissolved in deionized water (2.5 mL) under stirring at 298 K for 10 min. The reaction solution was allowed to stand at 298 K for 24 h to facilitate crosslinking. Then, the reaction mixture was crushed and soaked in 2 wt% aqueous NaBH₄ to reduce the formed imine bonds and unreacted aldehyde moieties. After leaving the reaction mixture in aqueous NaBH₄ for 24 h at 298 K, the resulting mixture was subjected to dialysis using a dialysis membrane tube with a molecular weight cutoff of 12,000 (Thermo Fisher Scientific, Waltham, MA, USA) against deionized water followed by lyophilization to achieve CTSG 1. CTSGs with different chitosan and OS concentrations were prepared using a similar method (Table 1).