Encapsulation of hemoglobin in a bicontinuous cubic phase lipid

The effects of encapsulating bovine hemoglobin (BHb) in the bicontinuous cubic phase formed by monooleoylglycerol and water was investigated with Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction. Cubic phase was formed in the presence of 1-10 wt% BHb. Studies using X-ray diffraction reveal that at 0.5-2.5 wt% BHb, the cubic phase structure is characterized by the double diamond lattice (Pn3m). At 2.5-5 wt% BHb, coexistence of two cubic phase structures, Pn3m and the gyroid lattice (Ia3d), was observed while at BHb concentrations higher than 5 wt% the gyroid structure persists. FTIR shows there is an increase in intensity of the free upsilon C = 0 (1745 cm(-1)) and a corresponding decrease in the intensity of the hydrogen bonded upsilon C = 0 (1720 cm(-1)) as the BHb concentration is increased. The upsilon C-O-CO peak shifts from 1183 cm(-1) to 1181 cm(-1) as the concentration of BHb is raised from 2.5 to 10 wt% indicating BHb may induce subtle changes in the interfacial region of cubic phase monoolein. The bandwidth of the upsilon(as)CH(2) stretch (2926 cm(-1)) increased in the presence of 5 wt% BI-IT, compared to samples with 2.5 or 10 wt% BHb. The increase in frequency of the upsilon(s)CH(2) stretch (2854 cm(-1)) induced by increasing temperature 20 to 60 degrees C was dampened when BHb was present compared to samples heated in isotonic buffer. Analysis of the amide I band at 1650 cm(-1) showed that the secondary structure of BHb is not affected by encapsulation in monoolein. In vitro release studies showed that 45% of the entrapped BHb was released after 144 h at 37 degrees C. The porous nature of bulk cubic phase was further demonstrated by diffusion of K2Fe(CN)(6) and conversion of 73% of the oxyhemoglobin to methemoglobin after 1 h. These results suggest that the cubic phase may be useful for encapsulation of Hb as a red cell substitute and for the encapsulation and delivery of other bioactive agents.