Dmx 360

Heparin was functionalized with methacrylate groups as described previously [14 (link)]. Briefly, 200 mg of sodium heparin (12 kDa; Smithfield Bioscience, Cincinnati, OH, USA) was first dissolved in 10 mL filtered distilled water (DW) to make a 2% (w/v) heparin solution. Then the solution was reacted with methacrylic anhydride (MA) in 10-fold molar excess (Sigma-Aldrich, St. Louis, MO, USA) for 12 h in the dark at 4 °C while maintaining the pH between 8 and 11 using 5 N or 1 N NaOH. The final product (Hep-MA) was precipitated in cold absolute ethanol with 10-fold volume (Sigma-Aldrich). After centrifugation at 5000g for 5 min at 4 °C, the supernatant was removed, and the precipitant was re-dissolved in 10 mL DW. To remove any unreacted reagents, Hep-MA was purified by dialysis against DW using a 3.5 kDa MW cut-off dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA) for 3 days. The resultant solution was freeze-dried (Sentry 2.0, SP Scientific, Warminster, PA, USA) for 3 days and stored at −20 °C for later use. The degree of methacrylation was determined by protein nuclear magnetic resonance (¹H NMR) spectroscopy (DMX 360; Bruker, Billerica, MA, USA) by dissolving 20 mg of Hep-MA in 600 μL deuterium oxide (D₂O) (Sigma-Aldrich).