Stability of Liposomal Membrane of Hemoglobin-Vesicles (Artificial Red Cells) for Over Years of Storage Evaluated Using Liquid Chromatography-Mass Spectrometry

Hemoglobin-Vesicles (Hb-V) are artificial oxygen carriers encapsulating a purified and concentrated Hb solution in liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), Cholesterol, 1,5-O-dihexadecyl-N-succinyl-l-glutamate (DHSG), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-poly(ethylene glycol) (PEG₅₀₀₀) (DSPE-PEG). The safety and efficacy of Hb-V have been studied extensively by both preclinical and clinical test methods. Deoxygenation of Hb-V prevents autoxidation of Hb and can extend its shelf life to 2 years at room temperature. However, the lipid components raise concerns about hydrolysis because Hb-V is dispersed in saline. For this study, we attempted to estimate the lipid degradation of long-term stored Hb-V using liquid chromatography-mass spectrometry. Analyses of lipid components extracted from the stored Hb-V showed that the degradation increased depending on the storage temperature. The calculated % remaining of intact lipids of Hb-V were 98.1% after 4 years and 90.4% after 7.2 years at 4 °C, 95.8% after 1 year and 86.7% after 2 years at 25 °C, and 85.6% after 6 months at 40 °C. The main degradation products were lyso-PC and palmitic acid which are hydrolyzed at the ester bond of DPPC. A few hydrolyzed products of DHSG and DSPE-PEG were also detected in Hb-V, but almost no degradation or oxidation products derived from Cholesterol could be identified. A shear test of Hb-V at 1500 s^-1 showed no significant increase in Hb leakage after storage of 2 years at 25 °C and 6 months at 40 °C. Lipid degradation products including free fatty acids would decrease the pH of the Hb-V dispersion and synergistically facilitate degradation, but it maintained pH 6.5 during 6 years at 4 °C, 2 years at 25 °C, and 3 months at 40 °C because of its high buffering capacity. These results indicate that the storage conditions for Hb-V are appropriate to minimize lipid degradation in the long term.