Fabrication of ORMs and catalase conjugation

Fabrication of oxygen-release microspheres
1.            Dissolve 250 mg of the synthesized polymer in 5 mL dichloromethane (DCM) to form a solution (oil phase).
2.            Dissolve 242 mg of polyvinylpyrrolidone (PVP) in 1 mL 30% H₂O₂ at 4°C overnight (inner water phase).
3.            Add the water phase rapidly into the oil phase.
4.            Sonicate by an ultrasonic liquid processor (Cole Parmer) using 30% of the maximum amplitude for 30 s to obtain the primary water-in-oil (W/O) emulsion.
5.            Pour the W/O emulsion into 50 mL poly(vinyl alcohol) (PVA) solution to form the water-in-oil-in-water (W/O/W) double emulsion.
6.            Stir for 3 hours to remove DCM.
7.            Centrifuge at 12000 x g for 15 min to collect microspheres.
8.            Wash the microspheres with DI water for 4 times to remove PVA.
9.            Freeze dry the microspheres, and store at -80^oC.

Conjugation of catalase onto oxygen-release microspheres  
1.            Weigh 40 mg of dry oxygen-release microsphere powder and add it into 6 mL, 5 mg/mL catalase solution (in DI water).
2.            Stir the mixture at 4°C for 4 hours.
3.            Centrifuge at 12000 x g for 15 min to collect microspheres.
4.            Wash with DI water for 3 times.
5.            Freeze dry the catalase-conjugated oxygen-release microspheres, and store at -80^oC.