Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95±0.02°C/min), medium (73±3°C/min) and fast (265±12°C/min) cooling rates and storage in liquid nitrogen, followed by a 37°C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%±5% vs 29%±4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.