Abstract
Although the disaccharide trehalose has been shown to protect cells during freezing and desiccation when present intracellularly, a major obstacle to using intracellular sugars for biopreservation applications is the impermeability of plasma membranes. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. Previous work has shown that the mechanism of red blood cell (RBC)–liposome interaction includes both liposome fusion with the RBC membranes, as well as tight adsorption of the vesicles onto the RBC surface. However, the fusion efficiency of liposomes was low, with only micromolar concentrations of trehalose delivered to the RBC cytosol. The purpose of this study is to enhance the efficacy of liposomes’ delivery of trehalose into RBCs with minimal detrimental effects on RBC membrane quality, by manipulating liposome physical properties and liposome–RBC incubation conditions. Charged and uncharged unilamellar liposomes were synthesized using an extrusion method to contain 300 mM trehalose in their aqueous core. Various concentrations of these liposomes (0.25–4.0 mM lipid) were incubated with RBCs at temperatures between 4°C and 37°C, for incubation times ranging from 15 min to 6 h. Red blood cell membrane quality was assessed using percent hemolysis, RBC morphology, phosphatidylserine (PS) and CD47 expression. The results of this study show that negatively charged liposomes containing PS in their membrane delivered about 100 times more trehalose (~15 mM) than neutral liposomes. Red blood cell–liposome incubation in an extracellular trehalose solution, as opposed to saline, results in a 34 ± 4 % increase of RBC intracellular trehalose concentration. In contrast to incubation time and temperature, liposome concentration and charge are not important predictors of RBC membrane injury. This study demonstrates that altering liposome physical properties and incubation conditions can cause an increase of RBC uptake of trehalose-containing liposomes, with minimal detrimental effects on RBC membrane quality.