title: Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality.
abstract: 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 (60:30:10 mol%) 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 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees 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.
authors.affiliation: Canadian Blood Services, Research and Development, 8249-114 Street, Edmonton, Canada AB T6G 2R8; Department of Laboratory Medicine and Pathology, University of Alberta, Canada.
authors.forename: Jelena L
authors.initials: JL
authors.lastname: Holovati
authors.affiliation: Canadian Blood Services, Research and Development, 8249-114 Street, Edmonton, Canada AB T6G 2R8; Department of Pathology, University of British Columbia, Canada.
authors.forename: Maria I C
authors.initials: MIC
authors.lastname: Gyongyossy-Issa
authors.affiliation: Canadian Blood Services, Research and Development, 8249-114 Street, Edmonton, Canada AB T6G 2R8; Department of Laboratory Medicine and Pathology, University of Alberta, Canada. Electronic address: jacker@ualberta.ca.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Acker
mesh_terms: D001793:Blood Preservation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects*; D006461:Hemolysis; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D010743:Phospholipids; D014199:Trehalose / Q000737:chemistry
chemical_list: D003451:Cryoprotective Agents; D004337:Drug Carriers; D008081:Liposomes; D010743:Phospholipids; D014199:Trehalose