112 Membrane changes in liposome-treated rat red blood cells upon hypothermic storage

Abstract

Liposomes, which are microscopic synthetic lipid vesicles, have been shown to improve red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42 day storage. Considering that the use of animal models is an essential step in preclinical studies for evaluating transfusion efficacy with novel manipulations of blood transfusion products, the aim of this study was to evaluate the effect of four liposome formulations on rat RBC membrane-related quality parameters during hypothermic storage. Unilamellar liposomes were synthesized using an extrusion method to contain lipid bilayer of saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):cholesterol, 7:3 mol%), unsaturated (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):cholesterol, 7:3 mol%), saturated charged (DPPC:cholesterol: phosphatidylserine (PS), 6:3:1 mol%) and unsaturated charged (DOPC:cholesterol:PS, 6:3:1 mol%) phospholipids. The leukoreduced, packed RBCs from Sprague-Dawley rats ( n = 3) were incubated for 1 h at 37 °C in a mixture of AS-3 solution with either HEPES-NaCl solution (control) or 2 mM lipid from four different liposome compositions. RBC membrane quality was assessed after treatment, one and five weeks of hypothermic storage by % hemolysis (Drabkins), deformability (ektacytometry), hematological indices (Coulter), morphology (microscopy), cholesterol and phospholipid concentration (colorimetry). All four liposome treatments resulted in significant decrease in percent hemolysis, with the effect most prominent with DOPC-treated RBCs (1.6 ± 0.1% vs. 3.1 ± 0.2 %, p = 0.05). No alterations in rat RBC membrane deformability, morphology, or hematological indices were detected in liposome treated RBCs immediately after the treatment. While DOPC liposome treatment resulted in significant increase in rat RBC phospholipid concentration, DPPC liposome treatment seemed to induce loss of phospholipids from the rat RBC membranes (0.30 ± 0.03 mg vs 0.15 ± 0.01 mg, p = 0.05). The cholesterol: phospholipid ratio in DOPC-treated rat RBCs was comparable to control (0.49 ± 0.06 vs 0.51 ± 0.08, p = 0.80). Upon the first week of storage DOPC-treated RBCs demonstrated significantly lower hemolysis (1.4 ± 0.1% vs. 2.5 ± 0.1%, p = 0.05) with values that are comparable to the ones of fresh RBC samples ( p = 0.25). All liposome treated RBCs continued to exhibit decreased hemolysis after 5 weeks of storage compared to the control ( p = 0.05), with uncharged formulations resulting in lower hemolysis than RBCs treated with charged liposomes (3.4 ± 0.2% vs 3.9 ± 0.4%, p = 0.01). The cell rigidity was significantly improved in DOPC-treated RBCs as demonstrated by the decreased KEI values compared to non-treated RBCs (1.01 ± 0.13 vs 1.36 ± 0.16, p = 0.05). The liposome treatment resulted in an improvement in rat RBC in vitro quality upon storage. The stabilizing effect was related to the chemical characteristics of each liposome formulation. The storage induced detrimental changes in the rat RBC membrane were minimized to a greater extent with the unsaturated than saturated liposome treatment. Also, negatively charged liposomes seemed to increase hemolysis upon storage. Therefore, unsaturated-uncharged liposomes will be used in the upcoming in vivo rat studies to further evaluate the pre-clinical effect of transfusing liposome-treated RBCs. Source of funding: Canadian Blood Services. Conflict of interest: None declared. jelena.holovati@ualberta.ca