The Effect of Liposome Treatment on the Quality of Hypothermically Stored Red Blood Cells

Abstract

Recent studies have demonstrated that liposome treatment of red blood cells (RBCs) leads to improved recovery and membrane integrity following cryopreservation protocols. However, the effect of liposome treatment on hypothermically stored RBCs has not been previously investigated. The current study has investigated whether liposome treatment could modify the membrane quality and deformability of hypothermically stored RBCs. Unilamellar liposomes were synthesized using an extrusion protocol. Three lipid bilayer compositions were investigated: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):PE:PS (8:1:1); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):PE:PS (8:1:1); and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):PE:PS (8:1:1). RBCs were treated with liposomes and subsequently stored for 42 days in HEPES-NaCl buffer and saline-adenine-glucose-mannitol. RBC quality was assessed by percent hemolysis, mean corpuscular volume (MCV), and RBC deformability (ektacytometry). DOPC and DMPC liposome treatment resulted in destabilization of the RBC membrane. Percent hemolysis values for DMPC-treated RBCs were higher than untreated controls throughout storage (P<0.05). DOPC-treated RBCs showed elevated levels of hemolysis compared to controls from day 21 of storage onward (P<0.05). In addition, DOPC and DMPC-treated RBCs were less deformable than untreated controls from days 21(P=0.02) and 14 (P<0.001) of storage onward respectively. [We suggest that these changes in RBC hemolysis and deformability are due to cholesterol extraction from the RBC membrane into the liposome fraction.] In contrast, DPPC-treated RBCs maintained hemolysis, MCV, and deformability values comparable to untreated controls. Future research addressing the optimal liposome composition for stabilizing the RBC membrane at cold temperatures could lead to effective strategies to combat the RBC membrane hypothermic storage lesion and ultimately improve the quality of hypothermically preserved blood.

Introduction

With the advent of modern preservative solutions and processing techniques, the shelf life of hypothermically stored red blood cells (RBCs) destined for transfusion has been extended to 42 days in Canada.^1,2 Although preservative solutions have addressed some components of the RBC “storage lesion” and have led to the improved overall quality of hypothermically stored RBCs, biochemical and biomechanical changes are still evident.¹ In addition, the transfusion of “older” RBC units has been associated with adverse clinical outcomes and increased morbidity and mortality in critically ill patients.^3,4 Despite the incremental advances that have occurred in the last 3 decades regarding our understanding of the RBC storage lesion and the development of new storage solutions, studies investigating more novel approaches to preserve RBC functionality during hypothermic storage are certainly warranted.

Many of the unaddressed effects of hypothermic storage lesion are manifested in the membrane of the RBC. It has been demonstrated that RBC hemorheology, specifically vascular adhesion and deformability, are impaired using current RBC hypothermic storage techniques.^5–7 Both of these hemorheological parameters are intimately related to the RBC membrane, suggesting that current hypothermic storage strategies fail to preserve normal RBC membrane function. A distortion in the normal asymmetric distribution of lipids in the membrane is observed, specifically the translocation of phosphatidyl serine from the inner to outer leaflet of the plasma membrane, which may lead to the premature removal of RBCs from the circulation once transfused.¹ In addition, microvesicles are shed from RBC membranes during storage, resulting in morphology changes as well as a decrease in the surface area to volume ratio, and the eventual build up of biologically active lipids and free hemoglobin in the storage supernatant.⁸ In the light of all these findings, it is apparent that new novel preservation strategies are needed to improve/modify the lipid component of the RBC membrane in the hope of improving the quality of stored RBCs.

The use of liposomes may be a possible method to modify the plasma membrane of hypothermically stored RBCs. Liposomes can be defined as synthetic microscopic vesicles composed of an intact lipid membrane surrounding an aqueous core. They are FDA approved as nonimmunogenic, nontoxic, and biocompatible carriers of various drugs, peptides, proteins and DNA which are suitable for clinical, diagnostic, and basic research purposes.^9–12 The synthetic nature of liposomes allows the researcher to prepare vesicles with specific phospholipid, cholesterol, and aqueous core compositions which has led to the use of liposomes in multiple disciplines, including cellular biopreservation applications. Numerous studies have utilized liposomes to study how sugars stabilize membranes during lyophilization and cryopreservation.^13–17 During lyophilization, RBCs have been shown to lose phospholipids through microvesiculation, resulting in deterioration of the RBC membrane and subsequent hemolysis.¹⁷ This membrane deterioration can be limited by the addition of small unilamellar liposomes to the lyophilization buffer.¹⁷ Interestingly, this type of membrane injury is reminiscent to the RBC storage lesion, suggesting that liposomes may be useful for improving RBC membrane integrity during hypothermic storage. Further, liposomes have been used as membrane models in cryobiology to study the protective mechanisms of permeating and nonpermeating cryoprotectants, as well as being utilized as membrane protectants themselves for the cryopreservation of bovine and equine sperm.^18–22 Although it has been established that the treatment of RBCs with liposomes results in improved membrane quality following cryopreservation and lyophilization, there have been no studies investigating the effect of liposomes on the quality of hypothermically stored RBCs. The current study has investigated whether the membrane quality and cellular deformability of RBCs could be modified with liposome treatment prior to routine hypothermic storage. Freshly isolated RBCs were treated with liposomes of varying lipid compositions (different lipid acyl chain lengths and degrees of saturation) and stored hypothermically for up to 42 days. Weekly assessments of RBC quality were performed including percent hemolysis, mean corpuscular volume (MCV), and RBC deformability analysis. The results of this study will be instrumental in determining if liposomal modification of RBC membranes is of any utility for the successful preservation of RBC membrane quality at hypothermic temperatures.

Materials and Methods

Liposome preparation

Liposomes were synthesized in the same manner as Holovati et al.,²³ with slight modifications. Dry 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was dissolved in chloroform (Alfa Aesar) and vigorously mixed with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) to a final molar ratio of 8:1:1 and a final total lipid concentration of ∼25 mM. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), as well as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were also prepared in an identical manner resulting in 8:1:1 molar ratios and final lipid concentrations of ∼25 mM. All lipids stocks were purchased from Avanti Polar Lipids and with the exception of dry DPPC, all utilized lipids were obtained predissolved in chloroform or chloroform:methanol mixtures. Multilamellar vesicles (MLVs) were produced by evaporating the chloroform:methanol solvents from the lipid solutions under dry nitrogen stream in a fume hood, lyophilized for ∼12 h (VirTis advantage Lyophilizer) and stored at −80°C until ready for hydration. All dry lipid cakes were hydrated with filter sterilized HEPES-NaCl buffer (pH 7.4, 274 mOsm). Following hydration, suspensions were frozen in liquid nitrogen, thawed in a 65°C water bath, and vortexed for 3 min for a total of 5 freeze-thaw cycles. The MLV suspensions were then subjected to an extrusion protocol to obtain a homogenous population of large unilamellar vesicles in a manner similar to Holovati et al.²³ Ten cycles of extrusion were performed for each liposome composition prepared. Liposome solutions were stored under nitrogen gas to minimize any lipid peroxidation and maintained at 1°C–6°C until further use for a maximum of 5 weeks. All liposome preparations will be referred to by the predominant lipid in each respective preparation (DPPC, DOPC, and DMPC).

Liposome characterization

Liposome diameter was determined using a Malvern 3000 HS Zetasizer (Malvern Instruments). Sizes are reported as the Z-average size (nm). A polydispersity value was also calculated which indicates the degree of size variability in the analyzed sample.²⁴ Polystyrene and silica sizing beads (200 and 400 nm) were used as controls for liposome size measurements (Bangs Laboratories, Inc.). The total lipid concentration of the liposome preparations (required to determine dose) was obtained by calculating the total phosphate content spectrophotometrically (Spectramax Plus; Molecular Devices) via the Fiske–Subbarow colorimetric assay as described previously.^23,25

RBC collection

Fresh RBCs (30 mL) were collected from healthy volunteers (n=6) in 7 mL vacutainer tubes containing K₃-EDTA anticoagulant (BD) using standard phlebotomy procedures. Following collection, the entire blood was centrifuged at 1500× g for 10 min at 4°C (Eppendorf Centrifuge 5810R) and the supernatant/buffy coat was removed by vacuum aspiration. The RBC pellets were then washed 3 times with HEPES-NaCl and counted on a hematology analyzer (Beckman Coulter AcT). The hematocrit was determined via microhematocrit centrifugation. All RBCs were utilized for liposome experiments within 4 h of collection.

Liposome incubation with RBCs

Each liposome formulation investigated was incubated with RBCs (12 mL, Hct≈5%) on a nutating mixer (VWR) for 1 h at 37°C immediately following RBC collection and processing. All liposome preparations were tested in duplicate for each of the 6 RBCs' donor samples. HEPES-NaCl buffer was used to dilute RBC samples and final concentrations of lipid were 2 mM for each respective liposome preparation. In addition, a small amount of saline-adenine-glucose-mannitol (SAGM; 2.0 mM adenine, 111.0 mM glucose, 41.2 nM mannitol, and 154.0 mM NaCl) was added to provide some nourishment for the RBCs (SAGM is a common additive solution used in RBC hypothermic preservation protocols). The ratio of SAGM to RBCs was comparable to that of packed RBCs units when hematocrit and sample volume were taken into account (275 μL of SAGM was added to 12 mL of RBCs with a hematocrit of 5%). Following incubation, liposomes were not removed from the storage solution. RBC samples were subsequently stored hypothermically at 1°C to 6°C for up to 42 days and quality assessments were performed at 1 week intervals throughout the hypothermic storage period.

RBC quality assessment

RBC quality was assessed by percent hemolysis, MCV, and deformability measurements. Percent hemolysis and MCV were determined using the cyanmethhemoglobin Drabkin's method²² and a hematology cell analyzer (Beckman Coulter AcT) respectively. RBC deformability was assessed as previously described by Stadnick et al.²⁶ In summary, RBCs were suspended in polyvinylpyrollidone and analyzed via ektacytometry (LORRCA) (Mechatronics) at shear stresses between 0.95 and 30 Pa. The resulting RBC deformability curve was transformed using an Eadie-Hofstee linearization technique, resulting in the deformability parameters EI_max (maximum RBC elongation) and K_EI (shear stress required to reach ½ of the EI_max).²⁶ EI_max and K_EI values were calculated for liposome-treated RBCs after each week of storage. RBC samples diluted and stored in HEPES-NaCl buffer and SAGM without liposome treatment served as controls. All EI_max and K_EI values are expressed as means±SEM.

Statistical analysis

The data were analyzed using commercial statistical software (SPSS Version 12.0, Lead Technologies) Due to the small sample size (n=6), nonparametric statistical analysis was performed. The Mann–Whitney U test was used to examine the differences in RBC quality parameters on a weekly basis. Unless specified, significant differences are expressed relative to age matched control RBC samples that are stored in HEPES-NaCl buffer and SAGM under the same conditions but without liposome incubation. Probabilities less than 0.05 were considered significant.

Results

The average diameter of the liposome preparations are shown in Table 1. The size distributions of all liposome preparations and polystyrene sizing beads were unimodal.

Table 1.

Sizes of Prepared Liposome Formulations

Sample	Z-average size (nm)	Polydispersity value
200 nm sizing beads	198	0.04
400 nm sizing beads	386	0.08
DPPC liposomes	153	0.04
DOPC liposomes	148	0.08
DMPC liposomes	152	0.06

All size distributions were unimodal.

DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine.

The percent hemolysis of RBCs increased in control and liposome-treated samples as storage duration increased (Fig. 1). RBCs treated with DMPC liposomes showed the most marked increase in percent hemolysis reaching a maximum of 53%±7% after 42 days of hypothermic storage (Fig. 1). At all time points, statistically significant increases in percent hemolysis were evident in DMPC liposome-treated RBCs. Although percent hemolysis in DOPC-liposome-treated samples remained similar to control values for the first 14 days of hypothermic storage, statistically significant increases were observed from day 21 of storage onward (P=0.03) (Fig. 1). By day 42, the percent hemolysis in DOPC-liposome-treated samples was comparable to DMPC liposome-treated samples (49%±7%). In contrast to DMPC and DOPC liposome-treated samples, DPPC liposome-treated sample showed percent hemolysis values similar to that of control RBCs (Fig. 1). Although there were no statistically significant decreases observed, percent hemolysis in DPPC liposome-treated RBCs tended to be slightly lower than untreated control RBCs from day 21 of hypothermic storage onward. DPPC liposome-treated RBCs exhibited significantly less hemolysis compared to DMPC liposome-treated samples at all time points and significantly less hemolysis compared to DOPC-treated samples from day 21 onward (Fig. 1). By 42 days of storage, hemolysis in DPPC-treated samples reached 12%±1% compared to 16%±1% in control samples which was not statistically significant (P=0.06).

FIG. 1.

Percent hemolysis of liposome-treated RBCs (DPPC, DOPC, and DMPC formulations) following hypothermic storage for up to 7 weeks (*P<0.05) (n=6). DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine; RBC, red blood cell.

MCV tended to increase during storage duration in both untreated control and liposome-treated samples (Fig. 2). At all sample points, DMPC liposome-treated RBCs had higher MCV values compared to untreated controls and DPPC/DOPC liposome-treated RBCs. This increase became statistically significant following 14 days of hypothermic storage (P=0.04) and remained significant throughout storage duration. There was no statistically significant difference in MCV observed between DPPC- and DOPC-treated samples or between DPPC/DOPC samples and untreated controls (Fig. 2).

FIG. 2.

MCV of RBCs following liposome treatment and subsequent hypothermic storage for up to 7 weeks (*P<0.05) (n=6). MCV, mean corpuscular volume.

RBC deformability deteriorated throughout storage duration for both untreated controls and liposome-treated samples (Figs. 3 and 4). A downward trend in EI_max was observed in all liposome-treated samples as well as untreated controls (Fig. 3). Statistically significant decreases in EI_max became evident in DMPC liposome-treated RBCs after 14 days of hypothermic storage (P<0.001) and 21 days of storage in DOPC liposome-treated RBCs (P=0.02). The decreases in EI_max observed in DOPC/DMPC liposome-treated samples remained statistically significant for the duration of storage (Fig. 3). In contrast to DOPC/DMPC liposome-treated RBCs, DPPC liposome treatment did not result in any significant differences compared to untreated control RBCs (Fig. 3). Two separate trends are visible in the K_EI values illustrated in Fig. 4. Following 7 days of hypothermic storage, an increase in K_EI was evident in all liposome-treated and untreated control samples. Subsequent to this, K_EI values in DPPC liposome-treated RBCs and untreated controls increased through storage (Fig. 4). In contrast, K_EI values of DMPC and DOPC liposome-treated RBCS dropped following 7 and 14 days of hypothermic storage respectively (Fig. 4). K_EI values of these 2 samples continued to decrease until day 28 where the K_EI values begin to increase for the last 2 weeks of hypothermic storage. Despite the visible trends, no statistically significant difference was detected in K_EI values between liposome-treated RBCs and untreated controls.

FIG. 3.

EI_max of RBCs following liposome treatment and subsequent hypothermic storage for up to 7 weeks (*P<0.05) (n=6).

FIG. 4.

K_EI of RBCs following liposome treatment and subsequent hypothermic storage for up to 7 weeks (n=6).

Discussion

Although liposomes have been previously used as membrane stabilizers in freeze-dried and cryopreserved RBCs,^16,17 this is the first study investigating the effect of liposomes on hypothermically stored RBCs. Liposome treatment of RBCs resulted in contrasting effects on RBC quality depending on liposome composition. DOPC and DMPC liposome treatment resulted in more drastic changes to RBC quality compared to DPPC liposomes, including greater percent hemolysis values and decreases in maximum deformability. In contrast, RBCs treated with DPPC liposomes showed slightly less hemolysis than untreated controls and retained deformation parameters that were similar in magnitude to that of untreated control RBCs after 42 days of hypothermic storage.

Due to the fact that the RBCs in this study are diluted and stored suboptimally in HEPES-NaCl buffer rather than citrate phosphate dextrose anticoagulant and SAGM additive solution, it is not surprising that hemolysis values are much higher than typically observed in blood banks.²⁷ However, storing RBCs under suboptimal conditions allowed us to more easily detect changes that liposome treatment had on RBC membrane quality. Our data shows that liposome treatment did not stabilize the membrane as indicated by the high percent hemolysis values in Fig. 1. The specific type of RBC-liposomal interaction occurring, which may include adsorption (“hemi-fusion”) to the RBC membrane with lipid transfer, complete membrane fusion, or no interaction, is likely to influence RBC membrane properties and hemolysis during hypothermic storage.²⁸ It has been previously reported that the depletion of RBC membrane cholesterol with the use of cyclodextrins induces RBC hemolysis, likely by removal of associated membrane components.^29,30 In addition, it has been shown that adsorption or hemi-fusion of liposomes often results in the depletion of membrane cholesterol in cells, and that this process is relatively fast.^31,32 Therefore, we believe that treatment with DMPC and DOPC liposomes in the current study resulted in an extraction of RBC membrane cholesterol and the subsequent disruption of membrane components, leading to eventual membrane failure and hemolysis. The apparent protective effect of DPPC liposomes, although not statistically significant, is more difficult to explain. A study by Stoll et al. demonstrated that DPPC liposomes interact very little with RBCs, therefore membrane protection via lipid transfer is unlikely.³³ However, since the liposomes in this study were not removed from the storage buffer following incubation, it is possible that DPPC liposomes may have had a coating effect on the RBC membranes, resulting in some sort of membrane protection. Further research is required to establish if DPPC liposomes are indeed protective against hemolysis during hypothermic storage, as well as what this protective mechanism may be.

Minimal effects on RBC MCV were observed in this study, as shown in Fig. 2. The gradual increase in MCV observed in all samples is an expected response, as decreasing ATP levels during storage eventually leads to the shutdown of membrane ion pumps (Na⁺/K⁺ ATPase and Ca⁺⁺ ATPase) which control the electrochemical gradient across the RBC membrane.³⁴ This results in the characteristic influx of water and cell swelling following storage for extended periods of time. The significantly higher MCV levels in DMPC-treated RBCs may be related to the higher levels of hemolysis observed, as the membrane status of these samples was clearly compromised.

Although the effect of liposomes on numerous RBC quality indicators has been previously investigated for cryopreserved RBCs, the deformability of liposome-treated RBCs has not yet been investigated.¹⁶ The results of this study demonstrate that the treatment and subsequent storage of RBCs with both DOPC and DMPC liposomes results in significant decreases in EI_max values, while DPPC treatment results in nearly no EI_max changes compared to control RBCs (Fig. 3). Considering that DOPC and DMPC liposome preparations are likely to result in cholesterol extraction from the RBC membrane, these findings are not unexpected. Changes in EI_max reflect alterations in the ultrastructure of the membrane or cytoskeleton matrix and numerous studies have investigated the effect of modifying the cholesterol content in RBC membranes.^29–31,35 Ohtani et al. demonstrated that the treatment of RBCs with cyclodextrins and subsequent extraction of cholesterol from the RBC membrane resulted in a discoid to sphereocyte morphology transformation.³⁰ Due to the decreases in the surface area to volume ratio associated with this morphology change, a decrease in maximal deformability is highly probable. The idea that cholesterol depletion is leading to the observed changes in RBC deformability is also supported by the K_EI values in Fig. 4. If cholesterol depletion is occurring during liposome incubation and subsequent storage, we would expect an increase in membrane fluidity as the phospholipids in the membrane will be less tightly packed.³⁶ This increase in membrane fluidity will result in less rigid membranes, which should be reflected in the K_EI values. K_EI values for DMPC and DOPC-treated RBCs exhibit an interesting, albeit nonstatistically significant decrease in K_EI values compared to that of both DPPC-treated and control RBCs for much of the storage duration. The extraction of cholesterol from RBC membranes is a logical explanation for this phenomenon.

Despite the apparent negative effects of DOPC and DMPC liposome preparations on the membrane quality of hypothermically stored RBCs, the results of this study are still encouraging. The protective effects of liposomes on cryopreserved RBCs reported by Holovati et al. occurred with cholesterol containing liposomes (DPPC:DPPS:Cholesterol in 60:10:30 mol% ratio).¹⁶ All liposome compositions investigated in the current study lacked cholesterol; therefore, the contrast in observed effects is not unexpected. If the depletion of membrane cholesterol is indeed the main factor contributing to RBC hemolysis and deformability deficiencies, it is likely this effect could be modified by adjusting the composition of the liposomes.¹¹ Stoll et al. demonstrated that inclusion of cholesterol into DLPC liposomes, subsequently incubated with RBCs, resulted in little depletion of RBC membrane cholesterol.³³ Therefore, if we can identify an appropriate ratio of cholesterol to lipid in prepared liposomes, it would be possible to deliver lipid to the RBC membrane without depleting RBC cholesterol to levels that result in membrane failure. By this rationale, it would then be possible to enhance RBC membrane fluidity via the delivery of lipids that are in the fluid phase at low temperatures. If the membrane fluidity of RBCs can be enhanced via liposome treatment without causing destabilization of the membrane, it may then be possible to decrease the membrane rigidity of hypothermically stored RBCs, resulting in the maintenance of RBC deformability during storage. More research is required to optimize the liposome composition that will elicit this effect and to investigate the function of such liposome-treated RBCs in an in vivo animal model.

Conclusion

The ability to systematically modify the membranes of RBCs could lead to future improvements in the quality of blood destined for transfusion. The results of this study demonstrate that liposomes have the capacity to alter membrane characteristics and specifically, the deformability of hypothermically stored RBCs differentially depending on the liposome lipid composition. DOPC and DMPC liposome treatment resulted in the greatest effect upon RBC membranes, while DPPC liposomes showed minimal interaction. The destabilizing effects on the RBC membrane exerted by DOPC and DMPC are likely related to the transfer of cholesterol from the RBC membrane to the liposome fraction. The apparent protective effects elicited by DPPC liposome treatment require further investigation. With future investigations into the optimal liposome lipid compositions for membrane stabilization of stored RBCs, the development of effective strategies to combat the membrane component of the RBC hypothermic storage lesion may be achievable.

Footnotes

Acknowledgments

The authors would like to thank Canadian Blood Services for funding and technical support as well as the Department of Laboratory Medicine and Pathology at the University of Alberta. Additionally, we would like to thank all the blood donors and the Network Centre for Applied Development (netCAD). This work was funded by grants from the Canadian Institute of Health Research and Canadian Blood Services. We have no conflicts of interest to declare.

Author Disclosure Statement

No competing financial interests exist.

References

Hess

. An update on solutions for red cell storage. Vox Sanguinis, 2006; 91:13–19.

Scott

, Lecak

, Acker

. Biopreservation of red blood cells: past, present, and future. Transfus Med Rev, 2005; 19:127–142.

, Sibbald

, Chin-Yee

. Effects of storage on efficacy of red cell transfusion: when is it not safe? Crit Care Med, 2003; 31:S687–S697.

Tinmouth

, Fergusson

, Yee

et al. Clinical consequences of red cell storage in the critically ill. Transfusion, 2006; 46:2014–2027.

Barshtein

, Manny

, Yedgar

. Circulatory risk in the transfusion of red blood cells with impaired flow properties induced by storage. Transfus Med Rev, 2011; 25:24–35.

Hanna

, Alexander

, Noga

et al. Blood banking–induced alteration of red blood cell flow properties. Transfusion, 2008; 48:136–146.

Mokken

, Kedaria

, Henny

et al. The clinical importance of erythrocyte deformability, a hemorrheolgical parmater. Ann Hematol, 1992; 64:113–122.

Hess

, Greenwalt

. Storage of red blood cells: new approaches. Transfus Med Rev, 2002; 16:283–295.

Banerjee

. Liposomes: applications in medicine. J Biomater Appl, 2001; 16:3–21.

10.

Chonn

, Cullis

. Recent advances in liposomal drug-delivery systems. Curr Opin Biotechnol, 1995; 6:698–708.

11.

Poste

, Papahadjopoulos

, Vail

. Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol, 1976; 14:33–71.

12.

Lasic

. Novel applications of liposomes. Trends Biotechnol, 1998; 16:307–321.

13.

Crowe

, Crowe

, Rudolph

et al. Preservation of freeze-dried liposomes by trehalose. Arch Biochem Biophys, 1985; 242:240–247.

14.

Crowe

, Womersley

, Crowe

et al. Prevention of fusion and leakage in freeze-dried liposomes by carboyhydrates. Biochim Biophys Acta, 1986; 861:131–140.

15.

Crowe

, Crowe

, Carpenter

et al. Interactions of sugars with membranes. Biochim Biophys Acta, 1988; 947:367–384.

16.

Holovati

, Gyongyossy-Issa

MIC

, Acker

. Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality. Cryobiology, 2009; 58:75–83.

17.

Kheirolomoom

, Satpathy

, Torok

et al. Phospholipid vesicles increase the survival of freeze-dried human red blood cells. Cryobiology, 2005; 51:290–305.

18.

Graham

, Foote

. Effect of several lipids, fatty acyl chain-length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing. Cryobiology, 1987; 24:42–52.

19.

Wilhelm

, Graham

, Squires

. Effects of phosphatidylserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior to and after cryopreservation. Cryobiology, 1996; 33:320–329.

20.

Anchordoguy

, Rudolph

, Carpenter

et al. Modes of interation of cryoprotectants with membrane phospholipids during freezing. Cryobiology, 1987; 24:324–331.

21.

Anchordoguy

, Cecchini

, Crowe

et al. Insights into the cryoprotective mechanism of dimethyl-sulfoxide for phospholipid-bilayers. Cryobiology, 1991; 28:467–473.

22.

Labbe

, Crowe

. Stability of the lipid component of trout sperm plasma membrane during freeze-thawing. Cryobiology, 1997; 34:176–182.

23.

Holovati

, Acker

. Spectrophotometric measurement of intraliposomal trehalose. Cryobiology, 2007; 55:98–107.

24.

Malvern Instruments Ltd. Zetasizer Nano Series User Manual, 1.1 2004 Worcestershire, UK: Malvern Instruments Ltd.

25.

Fiske

, Subbarow

. The colorimetric determination of phosphorus. J Biol Chem, 1925; 66:375–400.

26.

Stadnick

, Onell

, Acker

et al. Eadie-Hofstee analysis of red blood cell deformability. Clin Hemorheol Microcirc, 2010; 47:229–239.

27.

Zimrin

, Hess

. Current issues relating to the transfusion of stored red blood cells. Vox Sanguinis, 2009; 96:93–103.

28.

Holovati

, Gyongyossy-Issa

MIC

, Acker

. Investigating interactions of trehalose-containing liposomes with human red blood cells. Cell Preserv Technol, 2008; 6:131–142.

29.

Irie

, Otagiri

, Sunada

et al. Cycoldextrin-induced hemolysis and shape changes of human-erythrocytes in vitro. J Pharm Dyn, 1982; 5:741–744.

30.

Ohtani

, Irie

, Uekama

et al. Differential effects of alpha, beta, and gamma-cyclodextrins on human erythrocytes. Eur J Biochem, 1989; 186:17–22.

31.

Steck

, Ye

, Lange

. Probing red cell membrane cholesterol movement with cyclodextrin. Biophys J, 2002; 83:2118–2125.

32.

Steck

, Kezdy

, Lange

. An activation-collision mechanism for cholesterol transfer between membranes. J Biol Chem, 1988; 263:13023–13031.

33.

Stoll

, Stadnick

, Kollas

et al. Liposomes alter thermal phase behavior and composition of red blood cell membranes. Biochim Biophys Acta, 2011; 1808:474–481.

34.

Messana

, Ferroni

, Misiti

et al. Blood bank conditions and RBCs: the progressive loss of metabolic modulation. Transfusion, 2000; 40:353–360.

35.

Toyama

, Pais

, Meiselman

et al. Effects of cyclodextrins on RBC aggregation and blood viscosity. Clin Hemorheol Microcirc, 2007; 36:173–180.

36.

Richard

. Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells. J Supramol Struct, 1978; 8:413–430.