4.2. Synthesis of Hyaluronic Acid-Phospholipid Conjugate (HA-DPPE)

HA-DPPE conjugate was synthesized as described by Saadat et al. with minor modifications [19]. Briefly, 100 mg of HA 400 kDa were dissolved in 20 mL of MilliQ^® water and stirred until complete solubilization. Then, 2 M excess of EDC and NHS were added to the aqueous solution to activate the carboxylic groups of HA. The solution was stirred for 2 h at room temperature. DPPE (5 M excess of HA) was dissolved in 20 mL of tert-butanol/MilliQ^® water (9:1 v/v) in the presence of 0.1 mol triethylamine and mixed at 55 °C. The DPPE solution was then added dropwise to HA solution and the resulting mixture was stirred at 60 °C for 6 h followed by stirring at room temperature for additional 18 h. The mixture was dialyzed against MilliQ^® water using 3.5 kDa Spectra/Por dialysis bag for 48 h and then centrifuged twice at 1693 g for 30 min to completely remove, by precipitation, the free DPPE. Finally, the aqueous solution was lyophilized to get the solid HA-DPPE product. The reaction was monitored by thin layer chromatography (TLC) using F254 silica gel pre-coated sheets (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and a mixture of chloroform/methanol 70:30 v/v as mobile phase. TLC was visualized using molybdenum blue solution.