POLY(ETHYLENE GLYCOL)-INDUCED AND TEMPERATURE-DEPENDENT PHASE-SEPARATION IN FLUID BINARY PHOSPHOLIPID-MEMBRANES

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) ( PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of t he phospholipid polar headgroups. PEG (average molecular weight of 600 0 and in concentrations ranging from 5 to 20%, w/w) was added to the o utside of large unilamellar liposomes (LUVs). This leads to, in additi on to the dehydration of the outer monolayer, an osmotically driven wa ter outflow and shrinkage of liposomes. Under these conditions phase s eparation of the fluorescent lipid -(pyren-1-yl)]decanoyl-sn-glycero-3 -phosphocholine (PPDPC) embedded in various phosphatidylcholine matric es was observed, evident as an increase in the excimer-to-monomer fluo rescence intensity ratio (l(E)/l(M)). Enhanced segregation of the fluo rescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-s n-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-ph osphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphochol ine (POPC), for which temperature-induced phase segregation of the flu orescent lipid below T-m was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished ab ove characteristic temperatures T-0 well above their respective main p hase transition temperatures T-m. For 1,2-dipalmitoyl-sn-glycero-3-pho sphocholine (DPPC), DMPC, SOPC, and POPC, T-0 was observed at similar to 50, 32, 24, and 20 degrees C, respectively. Notably, the observed p hase separation of PPDPC cannot be accounted for the 1 degrees C incre ase in T-m for DMPC or for the increase by 0.5 degrees C for DPPC obse rved in the presence of 20% (w/w) PEG. At a given PEG concentration ma ximal increase in l(E)/l(M) (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1, 2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC)> DPPC> DMPC> SOPC> PO PC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycer o-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 199 4, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced d ehydration of liposomal membranes provides the driving force for the s egregation of the pyrene lipid. In brief, phase separation of PPDPC fr om the matrix lipid could be attributed to the diminishing effective s ize of the phosphatidylcholine polar headgroup resulting from its part ial dehydration by PEG. This in turn would allow for enhanced van der Waals interactions between the acyl chains of the matrix lipid, which then caused the exclusion of PPDPC due to the perturbing bulky pyrene moiety. Phase separation in DMPC/PPDPC liposomes was abolished by the inclusion of 25 mol % cholesterol and to a lesser extent by epicholest erol.