Synthesis and Characterization of Amphiphilic Hyaluronic Acid Polymers

Amphiphilic hyaluronic acid (HA) polymers were synthesized as described in previous reports.^{20 (link),26 (link)} Briefly, 40-45 mg HA (M_N = 10-20 kDa) was dissolved in 1:1 ultrapure water and DMF along with 30 mg of NHS and 30 mg of EDC. After mixing for 30 minutes to activate the HA carboxylic acid groups, 10 weight percent of hydrophobic reagent (octadecylamine or aminopropyl-pyrenebutanamide) was added to the HA solution and allowed to react for 24 hours. Samples were then removed and placed in 3500 MWCO dialysis tubing and dialyzed against 1:1 water and ethanol for 4 exchanges over 24 h, then against pure water for 8 exchanges over 48 h to remove any impurities. Finally, samples were frozen and freeze dried for later use. Structures of the obtained compounds were confirmed by ¹H NMR spectroscopy on a Bruker Advance 500 MHz NMR spectrometer at 25°C (Figure S1-S2). Samples were analyzed in DMSO-d₆ (99.9% D, Cambridge Isotope Laboratories). Data was processed in Mnova NMR (Escondido, CA). Hereafter, octadecyl-modified HA is referred to as ocdHA, and pyrene-butanamide substituted HA is referred to as pyHA.