Two distinct preparations of amphiphilic diblock copolymer vesicles (i.e. polymersomes), composed of (poly(ethylene oxide)-poly(butadiene)) (PEO-PBD), with molecular weights of 1.8 kDa and 10.4 kDa, offering different hydrophobic membrane thicknesses, were used to encapsulate the oxygen (O(2)) storage and transport protein hemoglobin (Hb) for possible application as a red blood cell (RBC) substitute. Key biophysical properties as well as the kinetics of polymersome encapsulated Hb (PEH) interaction with physiologically important gaseous ligands (O(2), carbon monoxide and nitric oxide) were measured as a function of the hydrophobic membrane thickness of the PEH particle. Taken together, the results of this work show that PEHs exhibit biophysical properties and retarded ligand binding/release kinetics (compared to cell-free Hb), which are similar to the behavior of RBCs. Therefore, PEHs have the potential to serve as safe and efficacious RBC substitutes for use in transfusion medicine.