Investigating interactions of trehalose-containing liposomes with human red blood cells

A major obstacle in using intracellular Sugars for the cryopreservation of mammalian cells is the inability of cells to synthesize or actively accumulate these Sugars. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. This Study examines the interactions of trehalose-containing liposomes with human red blood cells (RBCs). Unilamellar liposomes were synthesized using an extrusion method to contain trehalose in the aqueous core. Liposomal preparations were labeled with a lipophilic fluorophore rhodamine B chloride (R18) at quenching concentrations (4 mol%), or the 5(6)-carboxy fluorescein [5(6)-CF] marker to label the intraliposomal aqueous phase. Flow cytometry and fluorescent microscopy were used to assess the interactions between fluorescently labeled liposomes and RBCs. The delivery of liposomal contents into RBCs was assessed by spectrophotometric measurement of intracellular trehalose. The results of this study show that the transfer Of fluorescence from liposome to RBC population is clue, to both liposomal adsorption and fusion. The merger of membranes and lateral diffusion Of liposomal lipids into the RBC bilayer resulted in the spatial separation of membrane R18 fluorophores, and therefore, a dequenching effect, marked by a 344 +/- 11% increase of the RBC mean fluorescence intensity. The ability of 5(6)-CF and R18 liposomes to deliver their aqueous contents into RBCs, was confirmed by intracellular trehalose measurement. After treatment with 5(6)-CF liposomes, RBC trehalose concentration was 0.1.8 +/- 0.03 mM, whereas R18 liposomes delivered 0.96 +/- 0.07 mM trehalose. The results from this study strongly support the hypothesis that liposomes can be used as trehalose delivery vesicles. Delineating the mechanism of liposomal interaction with RBCs is an important step toward establishing the use of liposomes as tools for the intracellular delivery of stabilizing disaccharides for biopreservation purposes.