PT  - JOURNAL ARTICLE
AU  - Ming-Hao Li
AU  - Daniel P. Raleigh
AU  - Erwin London
TI  - Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage
AID  - 10.1101/2021.05.03.442494
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.03.442494
4099  - http://biorxiv.org/content/early/2021/05/04/2021.05.03.442494.short
4100  - http://biorxiv.org/content/early/2021/05/04/2021.05.03.442494.full
AB  - The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on the methyl-α-cyclodextrin catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is accomplished by pre-loading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at non-physiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological salt concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein.Competing Interest StatementThe authors have declared no competing interest.LUVlarge unidef-lamellar vesicleMLVmultilamellar vesiclesMαCDmethyl-α-cyclodextrinNBD-PC1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholinePBSphosphate-buffered salinePOPC1-palmitoyl-2-oleoyl-glycero-3-phosphocholinePOPE1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolaminePOPS1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt)TLCthin layer chromatographyTMADPH1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonatev/vvolume to volumew/wweight to weight